








key: cord-008613-tysyq6o4
authors: Thomas, Sheila M.; Lamb, Robert A.; Paterson, Reay G.
title: Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5
date: 1988-09-09
journal: Cell
DOI: 10.1016/s0092-8674(88)91285-8
sha: 
doc_id: 8613
cord_uid: tysyq6o4

The “P≓ gene of the paramyxovirus SV5 encodes two known proteins, P (M(r) ≈ 44,000) and V (M(r) ≈ 24,000). The complete nucleotide sequence of the “P≓ gene has been obtained and is found to contain two open reading frames, neither of which is large enough to encode the P protein. We have shown that the P and V proteins are translated from two mRNAs that differ by the presence of two nontemplated G residues in the P mRNA. These two additional nucleotides convert the two open reading frames to one of 392 amino acids. The P and V proteins are amino coterminal and have 164 amino acids in common. The unique C terminus of V consists of a cysteine-rich region that resembles a cysteine-rich metal binding domain. An open reading frame that contains this cysteine-rich region exists in all other paramyxovirus “P≓ gene sequences examined, which suggests that it may have important biological significance.

In recent years the catalog of mechanisms identified as having a role in the processing or modification of the initial RNA transcripts to yield mature mRNAs has increased markedly. In eukaryotic cells the most common process found is splicing of the primary RNA transcript (Padgett et al., 1986) . Less common variations on this splicing theme are alternative splicing, such that exons are excluded from some, but not all, of the mature mRNA, giving rise to sequence diversity in the encoded proteins (for review, see Breitbart et al., 1987) , and trans splicing, in which two independently transcribed RNAs are ligated to form the mature mRNA (Konarska et al., 1985; Solnick, 1985; Murphy et al., 1986; Sutton and Boothroyd, 1986; Krause and Hirsh, 1987; Koller et al., 1987) . Mechanisms of RNA transcript modification other than splicing include the phenomenon of RNA-editing in mitochondrial transcripts from trypanosomes, which is characterized by the presence in the mature mRNA of uridine residues that are not encoded in the gene (Benne et al., 1986; Feagin et al., 1987 Feagin et al., , 1988 Shaw et al., 1988) . In addition, a process related to RNA-editing is thought to occur in primary transcripts of the mammalian apolipoprotein-B gene, as two discrete mRNAs have been found, one of which has a U residue in place of a templated C (Powell et al., 1987; Chen et al., 1987) .

In animal virus infected cells, many examples of spliced and alternatively spliced viral mRNAs have been identified. In addition, an unusual cotranslational modification has been identified in vaccinia virus late transcripts that possess a poly(A) leader at the 5' end not encoded by the virus genome (Bertholet et al., 1987; Schwer et al., 1987) . The 5' poly(A) region is thought to be added by the vaccinia polymerase "stuttering" at a series of T residues on the template DNA (Schwer and Stunnenberg, 1988) . In many virus systems, in addition to modification of the primary RNA transcripts, the maximum protein coding potential on an mRNA is exploited by the use of alternative translation strategies that also have a potential role in the regulation of viral gene expression. Such mechanisms include translation from overlapping reading frames as observed for the coat, lysis, and synthetase proteins of bacteriophage MS2 (Atkins et al., 1979) and the adenovirus E1B proteins (Bos et al., 1981) ; ribosomal frameshifting that occurs to yield the gag-pol fusion proteins of Rous sarcoma virus, human immunodeficiency virus, or mouse mammary tumour virus (Jacks and Varmus, 1985; Jacks et al., 1987; Varmus, 1988) , and that also occurs in the polymerase encoding region of infectious bronchitis virus (Boursnell et al., 1987; Brierley et al., 1987) ; and the use of suppressor tRNAs to overcome translation termination during translation of the gag-pol fusion protein in Moloney murine leukemia virus (Yoshinaka et al., 1985) and the nsP4 protein of Sindbis virus (Strauss et al., 1983 )~ In negative strand RNA viruses, several of the processes discussed above are involved in the regulation of viral gene expression. For instance, in influenza A and B viruses, both spliced and unspliced mRNAs that are translated to yield polypeptides from overlapping reading frames have been identified (Lamb and Lai, 1980; Briedis and Lamb, 1982) , and influenza A viruses also provide an example of alternatively spliced mRNAs (Lamb et al., 1981) . Translation from overlapping reading frames on functionally bicistronic mRNAs has been shown to be the mechanism used to yield the NA and NB glycoproteins of influenza B virus (Shaw et al., 1983) and the P and C proteins of the paramyxoviruses Sendal virus and parainfluenza virus 3 and the morbillivirus, measles virus (Giorgi et al., 1983; Gupta and Kingsbury, 1985; Curran et al., 1986; Luk et al., 1986; Galinski et al., 1986; Spriggs and Collins, 1986; Bellini et al., 1985) .

Simian virus 5 (SV5), a prototype paramyxovirus, has a single-stranded, negative sense genomic RNA (vRNA) approximately 15,000 nucleotides in chain length that is transcribed in infected cells by the virion-associated RNA transcriptase to yield virus-specific mRNAs. The SV5 "P" gene has been shown to encode both the P protein (Mr = 44,000), and protein V (Mr = 24,000) by the arrest of translation in vitro of both P and V using a cDNA clone derived from SV5-specific mRNAs (Paterson et al., 1984) . In addition, the P and V proteins have been shown to have tryptic peptides in common, although no precursor-product kinetics could be demonstrated (Paterson et al., 1984; our unpublished data T66 CC6 6AC 6GG TTA 6CA ACA AGC 6AC TGC C66 TGC CAA CAB C6C AAT CCA CAA TCC ACA AT6 6AT CCC ACT 6AT CT8 ABC TTC TCC CCA BAT BAG ATC AAT AA6  0  N 

.120 .140 .160 .180 .200 CTC ATA GAS ACA G6C CTS AAT ACT GTA 6A6 TAT TTT ACT TCC CAA CAA 8TC ACA 66A ACA TCC TCT CTT 66A AA6 AAT ACA ATA CCA CC~ 666 6TC ACA 66A CTA 0 Leu-He~6~u~Thr- ,220

. .440

.460 .480 .500 .520 ACA TTA CCA TCA 66A TCC TAT ARE 666 6TT AA6 CTT GC8 AAA TTT 6GA AAA 6AA AAT CT6 AT6 ACA CG6 TTC ATC 6A6 6AA CCC AEA GAG ~T CCT ATC 6CA ACC 0 Thr- 

His-Gly-Ser-$er-Arg-AsD-Pro-61u-Arg-]le-Leu-Ser-61m-Pro-.540

.560 .580 .600 ,620 fiST TCC CCC ATC 6AT TTT AA6 A66 66C AG6 GAT ACC 66C 666 TTC CAT AGA A66 8A6 TAC TCA ATC 66A T66 6T8 66A GAT GAA 6TC AA6 6TC ACT SAG T66 TEE 0 Ser-~er-Pr~-~ze-A~P-Phe-Lys-Arq-6~y-Arg-As~-Thr-6~y~6~y-Phe-~is- ÷~ -Va~Pr~-~-~er-~e-Leu-~r~-6~y-A~a-6~y-He-Pr~-~|a-6|y-$er-I~e-6~u-6~y-Ser-Thr-6]~6er-As~-6~y- .660 .680 .700 .720 AAT CCA TCC TGT TCT CCA ATC ACC 6CT 6CA GCA AG6 CGA TTT GAA TGC ACT TGT CAC CAG TGT CCA 6TC ACT TGC TCT 6AA T6T 6AA CEA EAT ACT T 

.1280 .|298 ACT 6TT AT6 ACA CTG TAC TAA CCC T6A 666 TTT TA6 A Figure 1 . Nucleotide Sequence of Clone P203-1 in the mRNA Sense and the Predicted Amino Acid Sequence of the Two Open Reading Frames Nucleotide 1 is the 5'terminal nucleotide of the P and V mRNAs. After nucleotide 1298 there is a stretch of A residues in clone P203-1 (not shown) that is thought to represent part of the poly(A) tail on the mRNA. The amino acid numbering of the +1 reading frame is adjusted to conform with the residues predicted to exist in the P protein (see Figure 7B ). The sequence has been deposited in the EMBL/GenBank data base (accession no. J03142). and is part of the transcriptase complex (Buetti and Choppin, 1977) , while protein V is found in infected cells and is of unknown function (Peluso et al., 1977) .

The mechanism by which both the SV5 P and V proteins are encoded by a single gene has been investigated and we report here that P and V are amino coterminal proteins with different C-termini and are encoded by two separate mRNAs that differ by two nontemplated nucleotides.

To investigate the coding strategy used to express both the P and V proteins from a single gene on the SV5 virion RNA, the complete nucleotide sequence of three independently derived cDNA clones (P203-1, P10, and P127) was obtained using both dideoxynucleotide chain-terminating and chemical sequencing methods. The nucleotide sequence of clone P203-1 is presented in Figure 1 in the mRNA sense. It is 1298 nucleotides in length and contains an untranslated region of 60 nucleotides preceding the first AUG codon at nucleotides 61-63. Primer extension nucleotide sequencing on mRNA isolated from SV5infected cells indicated that nucleotide 1 is the 5' terminal nucleotide of the mRNA (data not shown). At the 3' end after nucleotide 1298 in the different clones, there is a stretch of A residues of variable length. The open reading frame following the first AUG codon (reading frame 0) is capable of encoding a protein of 222 amino acids. In the +1 reading frame there is an overlapping open reading frame of 250 amino acids, as illustrated schematically in Figure 2 . Although either reading frame could encode protein V (Mr "~ 24,000), neither of the open reading frames is apparently large enough to encode P, a protein of M r ~-44,000 (Paterson et al., 1984) , assuming the electrophoretic mobility of P is not aberrant. Examination of the predicted amino acid sequences indicates a region from residues 190-218 (29 amino acids) in the 0 reading frame that contains 7 cysteine residues, whereas the +1 reading frame only contains 1 cysteine at residue 357. Conversely, the +1 reading frame contains 6 methionine residues, whereas the 0 reading frame contains only one methionine, in addition to the initiation methionine.

To facilitate the elucidation of the coding strategy for P and V, we used monoclonal antibodies specific for the SV5 P and V proteins, which were generously made available by Dr. Rick Randall (Randall et al., 1987) . The P and V monoclonal antibodies have been assigned to three groups, members of which recognize three nonoverlapping antigenic sites (R. Randall, personal communication), here designated groups I, II, and III. The proteins immunoprecipitated from SV5 infected cell lysates by a representative member of each group are shown in Figure 3 (left section).

It can be seen that while P is immunoprecipitated by antibodies from all three groups, protein V is only recognized by the monoclonal antibody from group I. Recognition of both P and V by group I monoclonal antibodies indicates that they have amino acid sequences in common. The identity of the protein indicated by a star, migrating in a po- cysteine. Right section: Immunoprecipitation of SV5-infected cell lysates using the group I monoclonal antibody. It can be seen in this Immunoprecipitation that some NP coprecipitates with P and V using the group I antibody, probably because L, NP, P, and V exist in a complex (Randall et al., 1987;  our unpublished data). HN = hemagglutinin-neuraminidase protein, Mr ~--70,000; NP = nucleoprotein, Mr = 61,000; P = phosphoprotein, Mr = 44,000; M = matrix protein, Mr = 38,000; V = protein V, Mr = 24,000; * = polypeptide of unknown origin. Nucleotides Figure 4 . Antigenic Region Mapping of the P and V Monoclonal Antibodies on the P and V Gene Using T7 RNA Polymerase Runoff Transcripts, In Vitro Translation, and Immunoprecipitation The full-length P203-1 DNA insert cloned using Xbal linkers (XX) and a Hindlll to Xbal fragment (HX) containing the 3' two-thirds of the P203-1 DNA were placed under the control of the T7 promoter in the plasmid pGEM-2. The template DNA was linearized downstream of the T 7 promoter and the insert DNA by endonuclease digestion using EcoRI (XX and HX) or for truncated transcripts, by digestion of the full-length insert with Avail (XA) or Clal (XC), which cut at sites within the protein coding region. The runoff transcripts were translated in vitro using a rabbit reticulocyte lysate and the [3SS]methionine labeled proteins immunoprecipitated with the P specific monoclonal antibodies using the method described (Erickson and Blobel, 1979) .

(A) The immunoprecipitated in vitro translation products were analyzed by SDS-PAGE on 15% gels except for the 4 lanes at the far right, where a 10% polyacryiamide gel was used (see text). U = uninfected cell lysate labeled with Tran[35S]label; I = infected cell lysate labeled with Tran[35S]label as a marker lane; IV = proteins translated from poly(A)-containing mRNA isolated from SV5 infected cells; C = in vitro translation carried out in the absence of added mRNA; Gp I, Gp II, and Gp Ill indicate the monoclonal antibody used to immunoprecipitate the proteins observed in the region of the gel defined by the vertical lines either side of the antibody. Lanes XX = RNA transcribed from full-length cloned DNA; lanes HX = RNA transcribed from Hindlll to Xbal 3' two-thirds fragment; lanes XA = RNA transcribed from Xbal to Avail DNA fragment; lanes XC = RNA transcribed from Xbal to Clal DNA fragment. X = protein P, + = protein V, • = protein consistent in size with internal initiation at Met. 183 (210aa product in Figure 4B ), 1~ = protein consistent in size with internal initiation at Met. 233 (116aa product in Figure 4B ), 2~ = protein consistent in size with internal initiation at Met. 277, 3~ and 4~ = truncated products with protein synthesis initiated at Met. 1 (157aa and 98aa products respectively in Figure 4B ). (B) Schematic representation of the data accumulated in Figure 4A showing the protein products derived from the 0 and +1 reading frames, their relative number of amino acids assuming that all protein products from the +1 reading frame are initiated from internal methionine residues, the restriction endonuclease sites used in the generation of the RNAs, and a summary of the protein product reactivity with the Gp I, II, and III monoclonal antibodies.

sition between that of P and V, has not been investigated. To examine the relative ability by which the P and V proteins could be selectively radiolabeled, SV5-infected cells were labeled with either [35S]methionine or [35S]cysteine ( Figure 3 , middle section). In addition, cell lysates were immunoprecipitated with the group I antibody (Figure 3 , right section). As shown in Figure 3 , protein V was easily detected when labeled with either methionine or cysteine, whereas the P protein, although readily labeled with methionine, was poorly labeled with cysteine. These observations suggest that protein V possesses the cysteine-rich region encoded by the 0 reading frame, while the P protein apparently does not.

Coding

To define the regions of the nucleotide s e q u e n c e encoding the P and V proteins, we used the approach of making synthetic m R N A transcripts, translating the RNAs in vitro, and immunoprecipitating the products. The complete coding regions of clone P203-1 (nucleotides 44-1285) and a fragment containing the 3' two-thirds of the gene were subcloned into the transcription vector pGEM-2 such that they were under the control of the T7 R N A polymerase promoter. A series of mRNA-sense runoff transcripts were prepared as described in Experimental Procedures, translated in vitro using a rabbit reticulocyte lysate, and immunoprecipitated using the group I, II, and III monoclonal an-tibodies. The results obtained from such an assay are shown in Figure 4A and summarized in schematic form in Figure 4B . Interestingly, although the P protein could be translated in vitro using poly(A)-containing mRNAs isolated from SV5 infected cells ( Figure 4A , indicated as x in lanes IV), we were unable to detect the synthesis of the P protein when in vitro runoff transcripts were used to program the cell-free translation system. However, protein V was translated from both synthetic RNAs and mRNA was isolated from infected cells ( Figure 4A , indicated as +). Originally we thought it likely that frameshifting might be involved in the synthesis of P. However, because there is no detectable synthesis of the P protein when T7 runoff transcripts are used to program the rabbit reticulocyte lysate, whereas P is translated efficiently in vitro when poly(A) containing mRNA from infected cells is used, it would seem unlikely that ribosomal frameshifting is involved in the generation of the P protein. In addition to protein V, other in vitro synthesized proteins were observed, particularly during translation of the 3' two-thirds of the gene. The apparent size of the additional proteins is consistent with initiation of protein synthesis occurring at internal AUG codons in the +1 open reading frame: methionines 183 ( Figure 4A , closed circle), 233 ( Figure 4A , 1--*), and 277 ( Figure 4A, 2~) . From the sizes of the different protein products derived from the various runoff transcripts, it was possible to map unambiguously the regions on the open reading frames that were recognized by the monoclonal antibodies. In this way, monoclonal antibodies from group I were found to recognize the N-terminal region of the 0 open reading frame, group II antibodies to recognize a region from the N terminus of the +1 open reading frame, and group ill antibodies to recognize a C-terminal region of the +1 open reading frame.

In this analysis it should be noted that the largest in vitro translation product recognized by group II and III antibodies ( Figure 4A , indicated by a closed circle) was very similar in size to protein V (210 amino acids versus 221 amino acids) and had an almost identical electrophoretic mobility on SDS-PAGE. However, these polypeptides could be resolved when the samples were analyzed on a 10% polyacrylamide gel ( Figure 4A , right four lanes).

Thus, these data indicate that because P and V are immunoprecipitated by the Gp I monoclonal antibodies, and V cannot be immunoprecipitated by the Gp II and Gp III monocional antibodies, P and V are amino coterminal and protein V must be the product of the 0 open reading frame. In addition, these data indicate that protein P is derived from amino acid residues encoded by a large part of the +1 reading frame.

To obtain additional evidence that protein V is encoded by the 0 open reading frame and that the stop codon (TAA, nucleotides 727-729) that terminates translation in the 0 reading frame is not an artifact of the cDNA cloning procedure, we used an approach involving site-specific mutagenesis. If this stop codon in used to terminate translation of protein V, its elimination should prevent a normal-sized protein V from being synthesized, and a larger protein of 254 amino acids should be found. Nucleotides 727-729 (TAA) in the cloned DNA were changed to the triplet GCG (encoding alanine) as described in Experimental Procedures, the DNA containing the mutation was transcribed in vitro, and the resulting synthetic RNA translated in a rabbit reticulocyte lysate. As shown in Figure 5A , lanes 4 and 5, a protein (V*) larger than V ( Figure 5A , lane 3) that was recognized by the group I monoclonal antibody (Figure 5B, lanes 1 and 2) was synthesized.

As no evidence for frameshifting could be obtained, i.e., the inability to translate P in vitro from T 7 transcripts of P203-1 cloned DNA, the most plausible mechanism by which P and V are encoded is that a second mRNA species that is translated to yield the P protein exists. An insertion or deletion in such a mRNA would be expected to occur in the region of overlap between the two reading frames shown in Figure 2 . To search for the existence of a second mRNA population, nuclease $1 protection anal- (Lamb and Lai, 1982) ; F, control untreated probe; 0, no added mRNA; 1-100, increasing mRNA concentrations in the ratio 1:5:25:50:100. Numbers on the left of each panel are nucleotide sizes. A schematic diagram of the probe protected products, their nucleotide sizes, and the position of the uniquely labeled end, which is indicated by a star is shown beneath the autoradiograms.

ysis was performed using poly(A)-containing mRNA from SV5 infected cells and two DNA fragments from clone P203-1 that spanned the entire region of overlap, one uniquely 3' end-labeled at nucleotide 42 and the other uniquely 5' end-labeled at nucleotide 892. With each fragment, two nuclease $1 protected labeled fragments were detected, the full-length fragment used as a probe corresponding to a colinear mRNA transcript, and a smaller fragment present in 10%-20% abundance (data not shown). These data suggest that a second mRNA species exists that is derived from the P and V gene on the SV5 virion RNA and that it has a nuclease $1 sensitive site approximately between nucleotides 530 and 560. To define the location of this site more accurately, two shorter DNA fragments were used for the nuclease $1 analysis. One DNA fragment (n ucleotides 434 to 660) was 3' end-labeled at nucleotide 434 and the other DNA fragment (nucleotides 438 to 642) was 5' end-labeled at nucleotide 642. in addition to protection of the probe fragments corresponding to a colinear mRNA transcript, both probes protected smaller fragments found in 10%-20% abundance ( Figure  6 ). With both the 3' end and 5' end-labeled probes, smaller protected fragments (98 and 92 nucleotides respectively) increased in abundance with increasing concentrations of mRNA ( Figure 6 ). The size of the protected fragments mapped the region containing the nuclease Sl-sensitive region to between nucleotides 532 and 550.

A cDNA library derived from SV5-infected CV1 cell mRNAs (Paterson et al., 1984) was screened with an oligo-nucleotide probe to isolate cDNA clones specific for the P and V mRNAs. The nucleotide sequence of 22 P and V specific cDNA clones was obtained over the region of overlap between the 0 and +1 reading frames. In addition to cDNA clones having the same sequence as P203-1 (12 clones), a second population of cDNA clones was isolated (10 clones) that differed from P203-1 in containing two additional bases between nucleotides 548-551. The nucleotide sequence over the relevant region of a P mRNA clone and a V mRNA clone is shown in Figure 7A . it can be seen that whereas the V cDNA (P203-1) has four G residues between nucleotides 548 and 551, the P cDNA has six G residues (sections P and V, Figure 7A ). Although the simplest explanation of the nuclease $1 mapping data was that the P mRNA was a noncolinear transcript of the P and V gene containing an 18 nucleotide interrupted region, our retrospective explanation for the data is that nuclease $1 recognized a two nucleotide mismatch. The 3' break point maps precisely to the 4 G region in the V cDNA clone, while the 5' site did not. In addition to the inherent inaccuracies in measuring the precise size of DNA fragments, it is noted that the region 5' to the 4 G residues at nucleotides 548-551 is AT-rich and it may have been sensitive to digestion by the nuclease $1 (Hansen et al., 1981) . The two extra G residues cause a switch from the 0 reading frame to the +1 reading frame, and the predicted amino acid sequences are shown in Figure 7B . These data indicate that the P mRNA has the capacity to encode a polypeptide of 392 amino acids initiating at the AUG codon at nucleotides 61-63 and terminating at the TGA codon at nucleotides 1237-1239.

To determine whether the genomic virion RNA from which the SV5 mRNAs are transcribed contains four or six C residues complementary to the four or six G residues found in the two mRNAs, the sequence of the virion RNA (vRNA) was obtained as described in Experimental Procedures. As shown in Figure 7 (section vRNA), only a single cDNA sequence could be detected, and it contained four G residues complementary to four C residues in the vRNA.

To provide further evidence that the only difference between a P mRNA and a V mRNA is the presence of two nontemplated G residues, we investigated whether the P protein could be translated from a synthetic RNA derived from a P cDNA. To facilitate the genetic manipulation, an internal large restriction fragment spanning the region of interest in the P203-1 pGEM-2 vector was replaced with the comparable fragment from the P cDNA clone. In addition to using a "natural" P cDNA, we also changed the V cDNA clone P203-1 by site-specific mutagenesis to insert two additional G residues into the four G residues at nucleotides 548-551. Synthetic RNA was transcribed from both the "natural" and the "synthetic" P cDNA clones using T7 RNA polymerase, and translated in vitro in rabbit reticulocyte lysates. As shown in Figure 8 , both the "natural" and "synthetic" P cDNA clones yielded a P protein with an electrophoretic mobility identical to that of the P protein synthesized in infected cells and to the P protein translated in vitro from SV5-infected cell mRNA. All the P The nucleotide sequences of a P cDNA clone and a V cDNA clone in the region of nucleotides 541-564 are shown to illustrate the six G or four G residues in the P cDNA and V cDNA respectively. Sequencing was done by the chemical cleavage method (Maxam and Gilbert, 1980) . The sequence of the SV5 genomic template RNA (vRNA) is shown in the message sense as determined by dideoxy primer extension sequencing using reverse transcriptase (Air, 1979) . (B) The predicted amino acid sequence of the P and V proteins in the region of the six G or four G residues. Figure 8 . Expression of the P Protein from In Vitro Synthesized RNA A P cDNA clone was reconstructed in the P203-1 pGEM2 vector by replacing an internal large PstI-DNA fragment (nucleotides 225-660) with that from a P cDNA containing the two nontemplated G residues between nucleotides 548-551. The P203-1 DNA was also changed by site-specific mutagenesis to insert two additional G residues into the four G residues at nucleotides 548-551, and the mutated DNA subcloned into the pGEM-2 vector. RNA was transcribed with T7 RNA polymerase from both the "natural" and the "synthetic" P cDNA clones and translated in vitro using rabbit reticulocyte lysates. Lane 1 = SV5infected CV1 cell lysate as a marker. In vitro translated RNAs were as follows: lane 2 = no RNA control; lane 3 = poly(A)-containing mRNAs from SV5-infected CV1 cells; lanes 4 and 5 = "synthetic" P RNA from site-specifically mutated template DNAs; lane 6 = "natural" P RNA; lane 7 = V RNA from clone P203-1. Dashes = proteins P and V. Arrowhead and dot indicate protein products thought to originate from initiation at internal methionine residues 141 and 183 respectively.

proteins could be immunoprecipitated with the group I, II, and III monoclonal antibodies (data not shown). The protein products found in Figure 8 , lanes 4-6, indicated by an arrow and a dot, are thought to be internal initiation products from methionine residues 141 and 183 respectively. Protein V (Figure 8, lanes 3 and 7) is of a slightly different electrophoretic mobility from the smaller internal initiation product, and only protein V and not the internal initiation products are precipitated by the group I monoclonal antibodies (data not shown).

The finding of two extra G residues at a precise location in the P mRNA suggests that a signal would be needed to specify their addition, such as a region of strong secondary structure in the vRNA or mRNA. With the aid of the computer program FOLD (Intelligenetics Inc., Palo Alto, CA), the most stable secondary structure that can be predicted for nucleotides 520-620 of the P and V gene is one with an energy of •G = -53.7 kcal/mol and has the four templated C residues (nucleotides 548-551) immediately after a base-paired stem region (Figure 9 ). The Klenow fragment of E. coil DNA polymerase often yields artifactual sequencing bands at a run of several G residues when directly sequencing double-stranded DNA using the dideoxy chain-terminating method. As shown in Figure 9 the sequence of nucleotides 548-551 in the V clone (4 G residues) is easier to interpret than nucleotides 548-553 in the P clone (6 G residues). These artifactual bands can be eliminated when the sequencing is performed with a modified form of T7 DNA polymerase (Sequenase TM) in conjunction with dlTP instead of dGTP, unless there is a strong secondary structure in the template strand and then the artifacts are exacerbated (Tabor, 1987) . When this was done for the P clone DNA ( Left: The SV5 virion RNA sequence from nucleotides 520-620 of the P/V gene was examined for regions of strong secondary structure with the aid of the computer program FOLD (Inteiligenetics Inc., Pals Alto, CA). The stemloop structure shown has an energy of AG = -53.7 kcal/mol. The four C residues at nucleotides 548-551 are boxed. The arrow denotes the direction of mRNA transcription. Right: Nucleotide sequences obtained by the dideoxynucleotide chain-terminating method using the Klenow fragment of E. coil DNA polymerase or a modified form of T7 DNA polymerase (Sequenase TM) on a P clone cDNA template (Klenow and TT) or a V clone cDNA template (Klenow). In the Sequenase reactions (T7) dlTP was used in place of the usual dGTP. The region of the four or six G residues between nucleotides 548-551 is indicated by a star. 9) or V clone DNA (data not shown), the T7 DNA poiymerase nearly stopped its processive synthesis at nucleotides 543-550, which suggests that there is a native secondary structure in this region.

We have obtained the nucleotide sequence of the paramyxovirus SV5 P and V gene and have determined the strategy by which both proteins are expressed by a single gene. The P and V proteins are translated from two independent mRNAs that are synthesized in SV5 infected cells and are found to differ by the presence in the P mRNA of two additional nucleotides. A comparison of the nucleotide sequences of the P and V cDNAs and the SV5 genomic vRNA showed that the two additional G residues present in the P mRNA are not templated by the SV5 virion RNA (Figure 7 ). It could be argued that the vRNA sequencing might not detect a minor vRNA species of less than 5% abundance. However, there is no biological evidence for the involvement of more than one virus genome in the SV5 infectious cycle. Using a combination of in vitro translation of T7 runoff transcripts, immunoprecipitation of the in vitro synthesized proteins using monoclonal antibodies, oligonuc!eotide-directed mutagenesis, and metabolic labeling of SV5 infected cell proteins using specific amino acids ([35S]methionine or [35S]cysteine), we have shown that P and V are amino coterminal proteins that have different C-termini. The results presented here confirm earlier observations that the P and V proteins of SV5 have tryptic peptides in common (Paterson et al., 1984) . Thus SV5 differs from many paramyxoviruses and morbilliviruses that use functionally bicistronic mRNAs to synthesize the P protein, and a second protein known as C from overlapping reading frames (Giorgi et al., 1983; Galinski, et al., 1986; Bellini et al., 1985; Barrett et al., 1985) . Early peptide mapping data obtained for the P and "C-like" proteins of two other paramyxoviruses, Newcastle disease virus (NDV) and mumps virus, suggested that both proteins are encoded by the same reading frame (Collins et al., 1982; Herrler and Compans, 1982) . Recently the NDV and mumps virus P genes have been sequenced and found to contain one open reading frame (Sato et al., 1987; McGinnes et al., 1988; Takeuchi et al., 1988) from which it has been suggested that both the P and "C-like" proteins are derived, with the "C-like" protein arising from initiation at an internal AUG codon (McGinnes et al., 1988) . SV5 is therefore seemingly unique among paramyxoviruses in having two mRNAs transcribed from the P gene. The RNA-dependent RNA poiymerase of negative strand RNA viruses functions as part of a transcriptase complex composed of the template vRNA in tight association with the nucleoprotein (NP), and the P and L proteins, which are thought to be responsible for the polymerase activity (Buetti and Choppin, 1977; Hamaguchi et al., 1983) . Transcription of the virus-specific mRNAs by the transcriptase complex is believed to occur entirely in the cytoplasm of infected cells and is independent of host-cell mRNA synthesis.

The mechanism responsible for the addition of the untemplated G residues present in the P mRNA is unknown, nor is it known whether it is a cotranscriptional or posttranscriptional process. However, the virus-encoded RNA polymerase of negative strand RNA viruses is also responsible for the polyadenylation of virus-specific mRNAs, a process that is thought to occur by a "slippage" or "stuttering" mechanism involving the reiterative copying by the polymerase of a stretch of U residues located at the end of each gene. As the nontemplated G residues are added to the P transcript at a position where the template vRNA has a run of four C residues, it is possible that the SV5 polymerase "stutters" while copying this region of the genome and thus adds the nontemplated nucleotides. It is interesting that immediately upstream of the four C residues on the $V5 genomic RNA is the sequence The published nucleotide sequences of the P genes of several paramyxoviruses and the morbilivirus, measles virus, were translated in all three reading frames. In each case of a reading frame overlapping that for the P protein a cysteine-rich region was identified and is listed in the single letter amino acid code. Only the region of significant conservation of sequence is shown with its corresponding nucleotide number; the N-terminal region of the open reading frame is omitted. The star at the end of the amino acid sequence represents a translation termination codon. The boxes identify positions where three or more amino acids have been conserved in all six viruses, A dash indicates that a gap was placed in the alignment, and the star above the sequences shows the seven conserved cysteine residues. Sources for the P gene nucleotide sequences are as follows: SV5, this publication; mumps virus, Takeuchi et al., 1988; NDV, Sato et al., 1987; Sendai virus, Shioda et al., 1983 and Giorgi et al., 1983;  parainfluenza virus 3 (PI-3), Galinski et al., 1986, and Luk et al., 1986; measles virus, Bellini et al., 1985. 3'-AAAAUUCU-5' (Figure 9 ), which resembles the putative polyadenylation signal found at the end of SV5 genes and in fact is identical to the sequence at the end of the SV5 HN gene (Hiebert et al., 1985) , making this an attractive model for the mechanism by which the nontemplated Gs are added. However, it cannot be ruled out that the nontemplated G residues in the P mRNA are added as a consequence of some form of RNA-editing analogous to that found in mitochondrial transcripts in trypanosomes (Benne et al., 1986; Feagin et al., 1987 Feagin et al., , 1988 Shaw et al., 1988) or the mammalian apolipoprotein-B mRNA (Powell et al., 1987; Chen et al., 1987) . While screening the SV5 cDNA library for a P cDNA, 22 clones were sequenced across the region described above and only clones with either four or six G residues were found. This would suggest that whatever the mechanism involved in the addition of the nontemplated G residues, it is extremely specific. With the aid of the computer algorithm FOLD, a region of secondary structure was predicted for this part of the template RNA and it is therefore possible that this could play a role in either the mechanism itself or its regulation (Figure 9 ).

An examination of the predicted amino acid sequences of the P and V proteins reveals several interesting features. As mentioned above, the P and V proteins are amino coterminal and have their first 164 residues in common (Figure 1 ). An unusual feature of the shared region is the large number of proline residues; 17 prolines in 164 amino acids (Figure 1) . However, the most striking characteristic observed in either protein is the C-terminal portion of protein V, which consists of a cysteine-rich region bearing a remarkable resemblance to cysteine-rich regions found in the adenovirus E1A protein (for review see Moran and Matthews, 1987) , the yeast transcription factor GAL4 (Johnston and Dover, 1987) , and proteins belonging to the steroid hormone receptor superfamily (for review see Evans, 1988) . In these proteins and others possessing a similar domain it is thought that the binding of metal ions by the cysteine-rich region plays an important role in either the binding of nucleic acid by the protein, mediat-ing protein-protein interactions, or stabilizing oligomeric forms of a protein, as in the Tat protein of human immunodeficiency virus (Frankel et al., 1988) . Because of the significance of the cysteine-rich regions in other proteins, it was of interest to determine whether the sequence identified here in protein V had been conserved among other paramyxovirus P genes. Consequently, we compared the cysteine-rich region from protein V with the protein sequences predicted in all three reading frames from the nucleotide sequence of the P genes from mumps virus, NDV, Sendai virus, parainfluenza virus 3, and measles virus (Takeuchi et al., 1988; Sato et al., 1987; McGinnes et al., 1988; Shioda et al., 1983; Giorgio et al., 1983; Galinski et al., 1986; Luk et al., 1986; Bellini et al., 1985) . As shown in Figure 10 , a highly conserved cysteine-rich region was identified in an open reading frame in all the different paramyxovirus P gene sequences examined. Interestingly, the cysteine-rich region is more conserved between the different paramyxoviruses than is the amino acid sequence of the P protein encoded by the same nucleotides but translated in another reading frame (data not shown). As the P protein is part of the paramyxovirus transcriptase complex, the conservation of the cysteine-rich region must have important biological significance. It will be important to determine whether a protein containing this cysteine-rich region is synthesized in cells infected with other paramyxoviruses in addition to the already identified P or P and C proteins derived from the "P" gene.

The function of protein V has yet to be elucidated. However, as V is found associated with purified SV5 virions (our unpublished data) and as group I antibodies precipitate L, NP, P, and V in a complex (Randall et al., 1987; our unpublished data) , it remains a possibility that protein V may play a role in transcription and/or replication of the virus genome in infected cells.

Monolayer cultures of a variant of the MDBK line of bovine kidney cells and the TC7 clone of CV-1 cells were grown in Dulbecco's modified Eagle's medium (DmEm) supplemented with 10% fetal calf serum.

Stock virus was grown in MDBK cells infected with the W3 strain of SV5 (Choppin, 1964) as described previously (Peluso et al., 1977) . For all biochemical experiments, CV-1 cells were used and infected as described previously (Paterson et al., 1984) , except that for metabolic labeling of infected cell proteins, monelayers were incubated in methionine-and cysteine-free DmEm and proteins labeled using either Tran[35S]label (ICN Radiochemicals, Irvine, CA), 135S]cysteine or [35S]methionine (Amersham Corp., Arlington Heights, IL).

Messenger RNAs were isolated as described previously (Paterson et al., 1984) .

cDNA synthesis, isolation of SV5 specific clones, and the identification of cDNA encoding the various viral gene products has been described (Paterson et al., 1984) . Three clones, P10, P27, and P203-1 were sequenced over their entire length both by the chemical cleavage method (Maxam and Gilbert, 1980) and after subcloning into the Pstl site of the replicative form of bacteriophage M13mp19, by the dideoxy chain-termination method (Sanger et al., 1977) . Dideoxy primer extension sequencing on purified SV5 genomic RNA and poly(A)-containing mRNA was performed using avian myeloblastosis virus reverse transcriptase (Molecular Genetic Resources, Tampa, FL) and P gene specific primers as described previously (Air, 1979) . Direct sequencing of double-stranded plasmid DNA was carried out by the dideoxy chaintermination method using the Klenow fragment of E. coil DNA polymerase (Bethesda Research Laboratories, Gaithersberg, MD) as described by Sanger et al. (1977) or a modified form of T7 polymerase (Sequenase TM, United States Biochemical Corp., Cleveland, OH) according to the manufacturer's instructions. Restriction endonucleases, bacterial alkaline phosphatase, and T4 DNA ligase were obtained from Bethesda Research Laboratories, and T4 polynucleotide kinase from Pharmacia Fine Chemicals (Piscataway, NJ). Oligonucleotides were synthesized by the Northwestern University Biotechnology Facility on an Applied Biosystems (Foster City, CA) model 380B DNA synthesizer and were purified as described (Paterson and Lamb, 1987) .

The P203-1 cDNA was excised from pBR322 by Hhal and Mstll digestion, thereby eliminating the G/C tails introduced during cDNA cloning; Xbal linkers were added and the cDNA subcloned into the Xbai site of pGEM-2 (Promega Biotec, Madison, WI). Deletion of the 5' end of the gene was performed by digesting pGEM-2 containing the P203-1 cDNA with Xbai and Hindlll, isolating the 3' portion of the gene, addition of Xbal linkers, and subcloning back into pGEM-2. To construct both the protein V stop codon elimination mutant and the frameshift mutant, P203-1 cDNA was subcloned into the Xbal site of the replicative form of bacteriophage M13mp19, Oligonucleotide-directed mutagenesis was carried out according to the procedure of Zoller and Smith (1982) using mutagenic oligonucleotides consisting of 12 nucleotides either side of the site of the mutation. DNA containing the desired mutation was subcloned into pGEM-2 and the mutation verified by direct plasmid DNA sequencing using the dideoxy chain termination method (Sanger et al., 1977) and a P specific oligonucleotide primer.

For transcription of the entire coding region, plasmid DNAs were linearized downstream of the T7 promoter and the P or V insert, using EcoRI. For the synthesis of truncated forms of the rnRNA, the DNA template was linearized using either Avail or Clal, which recognize sites within the coding region of the cDNA. In vitro synthesis of mRNA was carried out as described previously (Hull et al., 1988) and 1 ~g of RNA was used to program a rabbit reticulocyte tysate as described below. T7 DNA-dependent RNA polymerase was obtained from Bethesda Research Laboratories, RNasin TM and RQ DNase TM from Promega Biotec, and 7r"G(5~)ppp(59G (sodium salt) was from Pharmacia Fine Chemicals.

In Vitro Translation of mRNAs mRNAs were translated in vitro using a micrococcal nuclease-treated rabbit reticulocyte lysate (Promega Biotec) according to the manufacturer's instructions. The in vitro-synthesized products were labeled using [35S]methionine. One-fifth volume of each translation reaction was immunoprecipitated as described below.

Immunoprecipitation was performed as previously described (Lamb et al., 1978; Erickson and Blobel, 1979) using monoclonal antibodies to the P and V proteins kindly provided by Dr. Rick Randall (Randall et al., 1987) . Samples were prepared for electrophoresis and analyzed by SDS-PAGE on 15% polyacrylamide gels as previously described (Lamb et al., 1978) .

Poly(A)-containing mRNAs from SV5 infected CV-1 cells were isolated as described (Paterson et al., 1984) . To determine whether more than one mRNA is transcribed from the P gene nuclease, Sl analysis was performed as previously described (Lamb and Lai, 1982) . The labeled DNA fragments used as probes were: a HhaI-Avall fragment and a BamHI-Pstl DNA fragment (nucleotides 42-889 and 434-660, respectively) 3' uniquely labeled at nucleotides 42 and 434, and a HhaI-Avall fragment and a BamHI-Hphl fragment (nucleotides 42-892 and 438-842, respectively) 5' uniquely labeled at nucleotides 892 and 642. Nuclease $1 was obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN.

Nucleotide sequence coding for the "signal peptide" and N terminus of the hemagglutinin from an Asian (H2N2) strain of influenza virus

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function

Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA

Measles virus P gene codes for two proteins

Major transcript of the frarneshift coxll gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA

Vaccin(a virus produces late mRNAs by discontinuous synthesis

The 2.2 kb Elb rnRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets

Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus

Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NSI and NS2 proteins

An efficient ribosome frarneshifting signal in the polymerase-encoding region of the coronavirus IBV

The transcriptase complex of the paramyxovirus SV5

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon

Multiplication of a myxovirus (SV5) with minimal cytopathic effects and without interference

Coding assignments of the five smaller m RNAs of Newcastle disease virus

Ribosomal initiation at alternate AUGs on the Sendal virus PIC mRNA

Early events in the biosynthesis of the lysosomal enzyme cathepsin

The steroid and thyroid hormone receptor superfamily

Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei

Developmentally regulated addition of nucleotides within the apocytochrome b transcripts in Trypanosoma brucei

Tat protein from human immunodeficiency virus forms a metal-linked dimer

Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins

Sendal virus contains overlapping genes expressed from a single mRNA

Translational modulation in vitro of a eukaryotic viral mRNA encoding overlapping genes: ribosome scanning and potential roles of conformational changes in the P/C mRNA of Sendal virus

Transcriptive complex of Newcastle disease virus. I. Both L and P proteins are required to constitute an active complex

T antigen repression of SV40 early transcription from two promoters

Synthesis of mumps virus polypeptides in infected vero cells

Hemagglutininneuraminidase protein of the paramyxovirus simian virus 5; nucleotide sequence of the mRNA predicts an N-terminal membrane anchor

Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus

Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting

Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumour virus gag-related polyproteins

Mutations that inactivate a yeast transcriptional regulatory protein cluster in an evolutionary conserved DNA binding domain

Evidence for in vivo trans splicing of pre-mRNAs in tobacco chloroplasts

Trans splicing of mRNA precursors in vitro

A trans-spliced leader sequence on actin mRNA in C. elegans

Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus

Spliced and unspliced messenger RNAs synthesized from cloned influenza virus M DNA in an SV40 vector: expression of the influenza virus membrane protein (M1)

Evidence for a ninth influenza viral polypeptide

Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins

Messenger RNA encoding the phosphoprotein (P) gene of human parainfluenza virus 3 is bicistronic

Sequencing end-labeled DNA with base-specific chemical cleavages

The P protein and the non-structural 38K and 29K proteins of Newcastle disease virus are derived from the same open reading frame

Multiple functional domains in the adenovirus E1A gene

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing

Splicing of messenger RNA precursors

Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor

Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5

Polypeptide synthesis in simian virus 5 infected cells

A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine

Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates

DNA sequencing with chain-terminating inhibitors

Molecular cloning and nucleotide sequence of P, M and F genes of Newcastle disease virus avirulent strain D26

Vaccinia virus late transcripts generated in vitro have a poly(A) head

Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5' poly(A) leader

A previously unrecognized influenza B virus glycoprotein from a bicistronic mRNA that also encodes the viral neuraminidase

Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons

Sequence of 3,687 nucleotides from the 3' end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P and C protein

Trans splicing of mRNA precursors

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins

Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon

Evidence for trans splicing in trypanosomes

SequenaseTM: step-by-step protocols for DNA sequencing with Sequenase TM. United States Biochemical Corporation

Molecular cloning and sequence analysis of mumps virus gene encoding the P protein: mumps virus P gene is monocistronic

Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon

Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment

We thank Margaret A. Shaughnessy for excellent technical assistance and Rick E. Randall of St. Andrews University, St. Andrews, Scotland for kindly providing the monoclonal antibodies to P and V. This research was supported by National Institutes of Health Research Grants AI-23173 and AI-20201. During the course of this work, R. A. L was an Established Investigator of the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 16 U.S.C. Section 1734 solely to indicate this fact.

