key: cord-339854-scb7pz87 authors: overend, christopher; yuan, lijuan; peccoud, jean title: the synthetic futures of vesicular stomatitis virus date: 2012-07-11 journal: trends biotechnol doi: 10.1016/j.tibtech.2012.06.002 sha: doc_id: 339854 cord_uid: scb7pz87 nan recent advances in synthetic biology have opened up the possibility of finely engineering viral genomes with the goal to understand and prevent viral diseases [1] . vesicular stomatitis virus (vsv) is one of the most promising viruses for engineering vaccines and oncolytic therapies [2] . its genome is able to accept large foreign sequences ( figure 1 ). vsv also remains viable as a pseudotyped virus (an enveloped virus expressing foreign glycoproteins in place of the native surface proteins) [3] . the broad tissue tropism allows for many different routes of administration, such as nasal sprays or intramuscular injection. vsv can be reliably grown to high titers in cell types approved for vaccine production. finally, negligible pre-existing immunity to this vector exists in the general population. protection against viral infections vsv-vectored vaccines have been explored as a strategy to protect against many emerging viral infections, exhibiting exceptional safety and efficacy. of particular interest is a study in which vsv expressing the h5 antigen from highly pathogenic avian influenza induced sterilizing immunity against heterologous challenge in mice [4] . in this experiment, it was impossible to detect challenge virus in lungs 3 days post-challenge. one study vaccinated and challenged macaques immunocompromised by simian-human immunodeficiency virus (shiv) infection [3] . the vaccine was well tolerated, and protected 67% of the vaccinated subjects from lethal zaire ebola virus (zebov) challenge. all unvaccinated animals succumbed by day 10 post-challenge. only the most severely immunocompromised vaccinated subjects succumbed to zebov challenge. this demonstrates the safety and efficacy potential of vsv when live virus vaccination would otherwise be contraindicated. although viral vectors are not traditionally utilized for vaccination against bacterial agents, they do have the potential for protection against intracellular bacteria. a significant level of protection has been reported against challenge with mycobacterium tuberculosis in mice 2 weeks post-vaccination [5] . this protection coincided with elevated but short-lived cd8 + t cells and interferon (ifn)-gproducing t cells following intramuscular injection. even more promising, recombinant vsv expressing a secreted form of a virulence factor protein for yersinia pestis, lcrv, induced high levels of lcrv-specific antibodies, protecting 90% of the mice challenged with 10 ld 50 [6] . protection against cancerous cells vsv is known to be exquisitely sensitive to ifn. many cancerous cells, unlike normal cells, are deficient in their ifn responses. therefore, it is possible for vsv to kill tumor cells, leaving healthy tissue at tumor margins unharmed. vsv is highly immunogenic, therefore, it can be detected and neutralized by the immune system before tumors are sufficiently destroyed. introducing the antiinflammatory glycoprotein from equine herpesvirus 1 into the vsv genome helped the virus to replicate more effectively in a rat model of hepatocellular carcinoma [7] . this resulted in a significant increase of the area of necrotic cells within the tumor. it also increased the survival of the animals. the safety of vsv has been demonstrated in immunocompromised macaques [3] . the neurovirulence observed in some vsv infections has been a source of concern. however, researchers have identified mutations erasing neurological signs [8, 9] . surprisingly, it has also been demonstrated that a replication-deficient vector may be a more potent vaccine than a replication-competent one [10] . this observation addresses many safety issues related to the use of replicating viruses. a more significant milestone is the start of the first clinical trials of vaccines derived from vsv. two clinical trials are recruiting healthy, hiv-free volunteers to examine the safety of potential hiv vaccines. these phase i studies will use either vsv expressing the hiv gag protein as a primary vaccine (clinicaltrials.gov identifier: nct01438606), or vsv-hiv gag as a booster to primary vaccination with dna expressing hiv gag and interleukin-12 as an adjuvant (clinicaltrials.gov identifier: nct01578889). successes of these trials will dramatically increase the viability of vsv derivatives in clinical applications. in light of promising successes in a wide variety of laboratory studies, it seems as though vsv will become a chassis of choice for engineering therapeutic viruses targeting a broad range of diseases. the genome is small enough to be manipulated in bacterial plasmids. its structure is simple enough to be modeled using computer assisted design software applications for synthetic biology. for instance, genocad [11] was used to generate the logical representations of the therapeutic viruses reviewed in this letter (figure 1 ). the coalescence of progress in synthetic biology similarly, a severe acute respiratory syndrome (sars) vaccine was designed by replacing the vsv g protein with the sars spike (s) protein (c). the avian influenza vaccine relies on the insertion of a sequence coding for the h5 antigen immediately next to the leader sequence (d). interestingly, the booster vaccine carries a different variant of the vsv g protein to escape the anti-vsv immune response acquired during the primary immunization. the tuberculosis vaccine relies on the insertion of an antigen between g and l (e). the yersinia pestis vaccine is structurally comparable to the avian influenza vaccine (f). finally, the anti-inflammatory oncolytic vsv has a large insert between g and l. the insert includes an anti-inflammatory glycoprotein (ehv1g) and a reporter gene (luc) separated by an internal ribosome entry site (ires) (g). these logical maps were generated using genocad [11] . trends in biotechnology october 2012, vol. 30, no. 10 synthetic viruses: a new opportunity to understand and prevent viral disease vesicular stomatitis virus: re-inventing the bullet vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates potent vesicular stomatitis virus-based avian influenza vaccines provide long-term sterilizing immunity against heterologous challenge heterologous boosting of recombinant adenoviral prime immunization with a novel vesicular stomatitis virus-vectored tuberculosis vaccine single-dose, virus-vectored vaccine protection against yersinia pestis challenge: cd4(+) cells are required at the time of challenge for optimal protection exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates peripheral immunization blocks lethal actions of vesicular stomatitis virus within the brain sars vaccine based on a replicationdefective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector writing dna with genocad (tm) all rights reserved this work was funded by national science foundation award ef-0850100 and a seed grant from the virginia tech institute for critical technologies and applied science. key: cord-345299-4k7qymqd authors: xiong, hua-long; cao, jia-li; shen, chen-guang; ma, jian; qiao, xiao-yang; shi, tian-shu; yang, yang; ge, sheng-xiang; zhang, jun; zhang, tian-ying; yuan, quan; xia, ning-shao title: several fda-approved drugs effectively inhibit sars-cov-2 infection in vitro date: 2020-06-05 journal: biorxiv doi: 10.1101/2020.06.05.135996 sha: doc_id: 345299 cord_uid: 4k7qymqd to identify drugs that are potentially used for the treatment of covid-19, the potency of 1403 fda-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic sars-cov-2 assay. four compounds, clomiphene (citrate), vortioxetine, vortioxetine (hydrobromide) and asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. the combination of clomiphene (citrate), vortioxetine and asenapine (hydrochloride) is much more potent than used alone, with ic50 of 0.34 μm. as of 26 may 2020, the covid-19 pandemic has claimed more than 342,000 lives, but yet effective drug is not available. it is time-consuming to develop vaccines or specific drugs for a disease caused by a novel defined virus like sars-cov-2. re-purposing of approved drugs may be a faster way to find treatment for covid-19. verification of drugs that might suppress sars-cov-2 by prediction, including drugs against similar virus and broad-spectrum antiviral agents (bsaas), is time-saving for drug re-purposing at the expense of missing some potential candidates. integrative, antiviral drug repurposing methods based on big data analysis or molecular docking and molecular dynamics are timesaving and high throughput. however, drugs identified by virtual screening still need to be verified in vitro and in vivo. in our previous research, a robust neutralization assay was established based on sars-cov-2 s-bearing vesicular stomatitis virus (vsv) pseudovirus and human ace2expressing bhk21 cells (bhk21-hace2) 1 . single-cycle infectious of recombinant vsv-sars-cov-2-sdel18 mimics the entry of sars-cov-2. the infection of pseudovirus can be detected by fluorescence 12 hours after infection, enabling the assay time-saving for high-throughput screening 1 . this pseudovirus based assay is suitable for screening drugs that can block the infection of sars-cov-2. in this study, the anti-sars-cov-2 potentiality of 1403 fda approved drugs were quantitatively evaluated by the pseudovirus-based assay. the screening procedure was illustrated in figure 1a and described in methods. the numbers of gfp-positive cells from drug treated wells were counted and divided by the number of infected cells from the well without treatment of drugs to calculate the relative value of infection rate. the results of two repetitions showed that most of drugs did not inhibit viral infection ( figure 1b ). fortyfour drugs with relatively better inhibitory effect were selected for further validation. in the second round of screening, inhibition of vsv-sars-cov-2-sdel18 virus infection and cell cytotoxicity were both detected (supplementary figure 1) . among them, 31 drugs were excluded due to cytotoxicity. 13 drugs were selected for analysis of specificity to vsv-sars-cov-2-sdel18 and verification by authentic sars-cov-2 assay. to verify whether these selected drugs act on spike protein of sars-cov-2 on the pseudovirus or the vsv backbone, we evaluated the inhibitory effect of these compounds on vsv-g (the sequence of gfp was inserted into the genome of vsv, so that the infection of vsv could be indicated by green fluorescence.). there were three compounds, including chloroquine diphosphate, ribavirin, and cetylpyridinium (chloride monohydrate), exhibited significant inhibitor effects on vsv-g, whereas no effect was noted for other compounds ( figure 1c ). including clomiphene (citrate), amiodarone (hydrochloride), vortioxetine, vortioxetine (hydrobromide) and asenapine (hydrochloride), were selected and the function of these compounds was confirmed using authentic sars-cov-2 assay ( figure 1d and e). among them, the inhibitory effects of clomiphene (citrate) and vortioxetine were comparable to chloroquine diphosphate, while vortioxetine (hydrobromide) and asenapine (hydrochloride) were slightly less effective. whereas amiodarone (hydrochloride) inhibited the infection of pseudovirus efficiently with ic50 around 4.44 μm, but it showed 5 no effect on authentic sars-cov-2 virus infection even used at a concentration of 100 μm. to further evaluate the potential of applying these drugs in prophylaxis and combination therapy, we treated the cell with pseudovirus and different drug combinations. the drug combinations were added either at the same time of pseudovirus infection or 6 hours pre-infection (table 1 vortioxetine is an antidepressant drug that is used to treat major depressive disorder in adults. vortioxetine was safe and well tolerated, it was approved in 2013 9 . so far, no previous study described its antiviral roles. it is reported that sever covid-19 patients have a high probability of suffering from mental illness. recently, another antidepressant drug fluvoxamine is evaluated for the potential to treat covid-19 by researchers from the washington university school of medicine, because the drug may prevent an overreaction 7 of the immune system called cytokine storms, which could result in life-threatening organ failure. the antiviral mechanism of vortioxetine remains unknow. however, it may bring physical and psychological benefits for covid-19 patients. asenapine is an antipsychotic medicine that is used to treat schizophrenia and bipolar i disorder and has been approved since 2009. notably, it showed less cytotoxicity in this study comparing to other drugs that could inhibit the infection of sars-cov-2. in summary, our study identified four fda-approved drugs that have the potential to suppress sars-cov-2 infection. the robust assay based on vsv-sars-cov-2-sdel18 pseudovirus screened out the potential drugs with high efficiency, then the inhibitory effect was confirmed by authentic sars-cov-2 assay. the inhibitory effect of vortioxetine and clomifene is superior and the mechanism of these drugs seems different from chloroquine. the combination of clomifene (citrate), vortioxetine and asenapine (hydrochloride) greatly decreases the ic50/ic90 of blocking virus infection. the clinical safety of these compounds has been evaluated and the availability of pharmacological data are expected to enable rapid preclinical and clinical evaluation for treatment of covid-19. based on the existing clinical results, it seems that it is difficult for one particular drug alone to significantly benefit covid-19 patients, and combination therapy is more likely to make the patient recover faster. this work identified novel drugs that suppress the infection of virus and provided more candidates for post-exposure prophylaxis and combination therapies. notice that no test in vivo has been conducted and the mechanism of these compounds also remains unknown. more researches are required to support the clinical application of these drugs for treatment of covid-19. vsv pseudovirus carrying truncated spike protein of sars-cov-2, named vsv-sars-cov-2-sdel18 virus, was packaged as previously described 1 . vsv-g was prepared in similar way 10 the relative value or inhibition rate of candidate drugs were calculated according to the decrease of gfp positive cell number (for pseudovirus-based assay) or cytopathic effect (for authentic sars-cov-2-based assay). the ic50 (the half maximal inhibitory concentration), ic90 (the concentration for the 90% of the maximum inhibition) and cc50 (the 50% cytotoxic concentration) values were calculated with non-linear regression, i.e. log (inhibitor) vs. normalized response -variable slope or log(agonist) vs. response-find ecanything using graphpad prism 7.00 (graphpad software, inc., san diego, ca, usa). 106.20 na "clo" means clomiphene (citrate), "vor" means vortioxetine, "ase" means asenapine (hydrochloride) and "cq" means chloroquine diphosphate. "pre-treatment" means cell was treated with drugs 6 hours before infection, while "cotreatment" means cells were treated with drugs at the time of infection. ic50, ic90 and cc50 were calculated using prism software (graphpad). "na" means the value can't be calculated. moi=0.1 robust neutralization assay based on sars-cov-2 s-bearing vesicular stomatitis virus (vsv) pseudovirus and ace2-overexpressed bhk21 cells triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial. the lancet %@ fda-approved selective estrogen receptor modulators inhibit ebola virus infection ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms clomiphene and its isomers block ebola virus particle entry and infection with similar potency: potential therapeutic implications characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the safety and tolerability of vortioxetine: analysis of data from randomized placebo-controlled trials and open-label extension studies generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines hua-long xiong, tian-ying zhang, quan yuan and ning-shao xia had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy the authors declare that they have no conflicts of interest. key: cord-316589-f1hq0xl5 authors: nagalo, bolni marius; breton, camilo ayala; zhou, yumei; arora, mansi; bogenberger, james m.; barro, oumar; steele, michael b.; jenks, nathan j.; baker, alexander t.; duda, dan g.; roberts, lewis rowland; russell, stephen j.; peng, kah whye; borad, mitesh j. title: oncolytic virus with attributes of vesicular stomatitis virus and measles virus in hepatobiliary and pancreatic cancers date: 2020-08-19 journal: mol ther oncolytics doi: 10.1016/j.omto.2020.08.007 sha: doc_id: 316589 cord_uid: f1hq0xl5 abstract recombinant vesicular stomatitis virus (vsv)-fusion and hemagglutinin (fh) was developed by substituting the promiscuous vsv-g glycoprotein (g) gene in the backbone of vsv with genes encoding for the measles virus envelope proteins f and h. hybrid vsv-fh exhibited a multifaceted mechanism of cancer-cell killing, and improved neurotolerability over parental vsv in preclinical studies. in this study, we evaluated vsv-fh in vitro and in vivo in models of hepatobiliary and pancreatic cancers. our results indicate that high intrahepatic doses of vsv-fh did not result in any significant toxicity and were well tolerated by transgenic mice expressing the measles virus receptor cd46. furthermore, single intratumoral treatments with vsv-fh yielded improved survival and complete tumor regressions in a proportion of mice in the hep3b hepatocellular carcinoma model, but not in mice xenografted with bxpc3 pancreatic cancer cells. our preliminary findings indicate that vsv-fh can induce potent oncolysis in hepatocellular and pancreatic cancer cell lines with concordant results in vivo in hepatocellular cancer and discordant in pancreatic cancer, without the vsv-mediated toxic effects previously observed in laboratory animals. further study of vsv-fh as an oncolytic virotherapy is warranted in hepatocellular carcinoma and pancreatic cancer, to understand broader applicability and mechanisms of sensitivity and resistance. vehicles. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] however, successful clinical translation of vsv has been hindered by a number of factors, including neurovirulence, liver toxicity, and variable sensitivity of malignant tumors to vsv oncotherapy. 3, 4, [13] [14] [15] [16] [17] [18] several studies have attempted to remedy these limitations, with varying degrees of success. [19] [20] [21] however, additional investigations and new approaches are needed before vsv-based virotherapy can be used clinically to treat human cancers. able to cross the human blood-brain barrier and propagate in the brain, treatment with vsv-fh will allow for mitigation of neurotoxicity because most normal neuronal cells lack surface expression of cd46, [27] [28] [29] [30] which vsv-fh uses for cellular entry by virtue of its oncolytic mv attribute. 22, 25, 26 vsv-fh has a robust and rapid lytic cycle attributable to the vsv platform and a preferential tropism for cd46-overexpressed cells from the mv platform. cd46 is ubiquitously expressed in all nucleated cells; however, its overexpression has been reported in human tumor cells and tissues, including hpbcs. [31] [32] [33] [34] [35] [36] [37] we have previously shown that hybrid vsv-fh mediates a higher degree of human tumor selectivity, which is strongly associated with the number of cd46 molecules expressed on target cells. 26 we also demonstrated that the multifaceted mechanisms of cancer-cell killing by vsv-fh are enhanced because of its inherent fusogenic activity. 25, 26 furthermore, we showed that treatment with vsv-fh has the added benefit of being well-tolerated and less neurotoxic in laboratory rodents than parental wild-type (wt)-vsv, which causes severe encephalitis in laboratory animals. 25, 26 hpbcs have extremely high rates of mortality and are increasing in incidence. 38 hbpcs. as previously described, the oncolytic virus vsv-fh-enhanced green fluorescent protein (egfp) was generated by substitution of the vsv to further investigate the difference in the infectivity and cpe of to determine whether vsv-fh is causally associated with liver damage and liver toxicity in animal models. analyses of multiple toxicology parameters, including complete blood counts (cbc), serum chemistry, and liver function tests, were performed at 2, 21, and 45 days after treatment. as shown in figure although body weight consistently increased from 4 days after injection until the end of the study ( figure 4b ). as shown in figure collectively, these data indicate that intrahepatic injection of vsv-fh did not elicit significant liver toxicity in the treated animals (100% survival) or result in impairment (kaplan-meier method, p<.05). hep3b ( hep3b cohort resulted in a significant difference in overall survival (p=.01 [ figure 5c ]), indicating effectiveness of treatment with vsv-fh. in this study, we evaluated whether treatment with oncolytic vsv-fh could trigger a potent cytotoxicity effect in hbpc cell lines in vitro and in vivo using animal models. we conducted a causality assessment in the context of potential vsv-fh-induced liver damage and toxicity in a human cd46 transgenic mouse model, expressing murine interferon alpha receptor (cd46 ifnαr wt/wt). this particular mouse strain has been shown to be susceptible to infections by viruses that use human cd46 as entry receptor, including measles virus, vsv-fh and some adenoviruses. 30,45 46 additionally, we compared cytotoxic effects of vsv-fh with that of another recombinant, neuro-attenuated vsv that is currently being evaluated in a clinical trial (vsv-hifnβ-nis). 14 in this study, we showed that vsv-fh high intrahepatic doses of vsv-fh in healthy transgenic mice expressing human cd46 were found to be manageable, safe, and tolerable. there was a no statistically significant increase in liver enzyme levels (alt, ast), granulocytes, and total bilirubin at the end of study, but not in alkaline phosphatase. in the absence of severe toxic effects, we deduced that these increases may not result in clinically significant sequelae and could have been confounded by blood hemolysis from the terminal cardiac puncture, which is known to interfere with the measurement of liver vsv-fh-egfp was used to infect 5×10 5 to determine the production of vsv-fh infectious particles, for cell viability assays, 1. under a mayo clinic institutional animal care and use committee-approved protocol, we conducted the following in vivo evaluations. to assess potential toxicity, especially hepatotoxicity, 30 recent advances in vesicular stomatitis virus-based oncolytic virotherapy: a 5-year update oncolytic recombinant vesicular stomatitis virus (vsv) is nonpathogenic and nontransmissible in pigs, a natural host of vsv safety studies in tumor and non-tumor-bearing mice in support of clinical trials using oncolytic vsv-ifnbeta-nis a novel chimeric oncolytic virus vector for improved safety and efficacy as a platform for the treatment of hepatocellular carcinoma pre-clinical development of a vaccine against lassa fever a vsv-based zika virus vaccine protects mice from lethal challenge long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsvbased vaccine the oncolytic virus vsv-gp is effective against malignant melanoma a recombinant vsv-vectored mers-cov vaccine induces neutralizing antibody and t cell responses in rhesus monkeys after single dose immunization enhanced safety and efficacy of oncolytic vsv therapy by combination with t cell receptor transgenic t cells as carriers comparative oncology evaluation of intravenous recombinant oncolytic vesicular stomatitis virus therapy in spontaneous canine cancer the vesicular stomatitis virus-based ebola virus vaccine: from concept to clinical trials re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency potent systemic therapy of multiple myeloma utilizing oncolytic vesicular stomatitis virus coding for interferon-beta neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates a human case of encephalitis associated with vesicular stomatitis virus (indiana serotype) infection. am safety studies on intrahepatic or intratumoral injection of oncolytic vesicular stomatitis virus expressing interferon-beta in rodents and nonhuman primates relative neurotropism of a recombinant rhabdovirus expressing a green fluorescent envelope glycoprotein vesiculovirus neutralization by natural igm and complement characteristics of oncolytic vesicular stomatitis virus displaying tumor-targeting ligands rvsv(m delta 51)-m3 is an effective and safe oncolytic virus for cancer therapy retargeting vesicular stomatitis virus using measles virus envelope glycoproteins. hum lassa-vesicular stomatitis chimeric virus safely destroys brain tumors cd30-targeted oncolytic viruses as novel therapeutic approach against classical hodgkin lymphoma oncolytic potency of her-2 retargeted vsv-fh hybrid viruses: the role of receptor ligand affinity faster replication and higher expression levels of viral glycoproteins give the vesicular stomatitis virus/measles virus hybrid vsv-fh a growth advantage over measles virus nectin 4 is the epithelial cell receptor for measles virus tumor cell marker pvrl4 (nectin 4) is an epithelial cell receptor for measles virus measles virus spread between neurons requires cell contact but not cd46 expression, syncytium formation, or extracellular virus production a matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain high cd46 receptor density determines preferential killing of tumor cells by oncolytic measles virus systematic immunohistochemical analysis of the expression of cd46, cd55, and cd59 in colon cancer antibody-drug conjugate targeting cd46 eliminates multiple myeloma cells rna interference characterization of proteins discovered by proteomic analysis of pancreatic cancer reveals function in cell growth and survival p53 regulates cd46 expression and measles virus infection in myeloma cells bioinformatic analysis of the membrane cofactor protein cd46 and microrna expression in hepatocellular carcinoma cell-surface density of complement restriction factors (cd46, cd55, and cd59): oral squamous cell carcinoma versus other solid tumors. oral surg oral med oral pathol oral radiol endod 103 projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the united states pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment and outcomes systemic treatment options in hepatocellular carcinoma amalgamating oncolytic viruses to enhance their safety, consolidate their killing mechanisms, and accelerate their spread oncolytic measles viruses displaying a single-chain antibody against cd38, a myeloma cell marker preclinical pharmacology and toxicology of intravenous mv-nis, an oncolytic measles virus administered with or without cyclophosphamide roles of macrophages in measles virus infection of genetically modified mice vaccines within vaccines: the use of adenovirus types 4 and 7 as influenza vaccine vectors vesicular stomatitis virus expressing interferon-beta is oncolytic and promotes antitumor immune responses in a syngeneic murine model of non-small cell lung cancer vascularization, oxygenation, and the effect of sunitinib treatment in pancreatic ductal adenocarcinoma xenografts role of the tumor microenvironment in pancreatic cancer antitumor effect of angiotensin ii type 1 receptor blocker losartan for orthotopic rat pancreatic adenocarcinoma hyaluronidase expression by an oncolytic adenovirus enhances its intratumoral spread and suppresses tumor growth key: cord-286390-ytgw3j4s authors: case, james brett; rothlauf, paul w.; chen, rita e.; liu, zhuoming; zhao, haiyan; kim, arthur s.; bloyet, louis-marie; zeng, qiru; tahan, stephen; droit, lindsay; ilagan, ma. xenia g.; tartell, michael a.; amarasinghe, gaya; henderson, jeffrey p.; miersch, shane; ustav, mart; sidhu, sachdev; virgin, herbert w.; wang, david; ding, siyuan; corti, davide; theel, elitza s.; fremont, daved h.; diamond, michael s.; whelan, sean p.j. title: neutralizing antibody and soluble ace2 inhibition of a replication-competent vsv-sars-cov-2 and a clinical isolate of sars-cov-2. date: 2020-07-03 journal: cell host microbe doi: 10.1016/j.chom.2020.06.021 sha: doc_id: 286390 cord_uid: ytgw3j4s abstract antibody-based interventions against sars-cov-2 could limit morbidity, mortality, and possibly transmission. an anticipated correlate of such countermeasures is the level of neutralizing antibodies against the sars-cov-2 spike protein, which engages with host ace2 receptor for entry. using an infectious molecular clone of vesicular stomatitis virus (vsv) expressing egfp as a marker of infection, we replaced the glycoprotein gene (g) with the spike protein of sars-cov-2 (vsv-egfp-sars-cov-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. we also developed a focus-reduction neutralization test with a clinical isolate of sars-cov-2 at biosafety level 3. comparing the neutralizing activities of various antibodies and ace2-fc soluble decoy protein in both assays revealed a high degree of concordance. these assays will help define correlates of protection for antibody-based countermeasures and vaccines against sars-cov-2. additionally, replication-competent vsv-egfp-sars-cov-2 provides a tool for testing inhibitors of sars-cov-2 mediated entry under reduced biosafety containment. here, we developed a simple and robust bsl2 assay for evaluating sars-cov-2 entry 82 and its inhibition by antibodies. we engineered an infectious molecular clone of vesicular 83 stomatitis virus (vsv) to encode the sars-cov-2 s protein in place of the native envelope 84 glycoprotein (g) and rescued an autonomously replication-competent virus bearing the spike. through passage of vsv-egfp-sars-cov-2, we selected a gain-of-function mutation in s that 86 allowed more efficient viral propagation yielding titers of > s aa as determined by expression of the virus-encoded egfp reporter (fig 1a, right panel) . however, vsv-egfp-sars-cov-2-s aa propagation was inefficient on vero ccl81 cells. this 106 result prompted us to test additional modifications of the cytoplasmic tail of s, which also were 107 defective in autonomous amplification (fig s1b) . to overcome this limitation, we used a forward 108 genetic approach to isolate two adaptive variants of vsv-egfp-sars-cov-2-s aa (fig s1c) . which truncates the cytoplasmic tail of sars-cov-2 s by 21 residues (fig 1a) . this virus, hereafter referred to as vsv-sars-cov-2-s δ21 , was passed 12 times in total to assess genetic 6 stability by next generation sequencing, which revealed no additional mutations in the spike 118 (sra: srr11878607; bioproject: prjna635934). comparison of plaque morphology of vsvsars-cov-2-s δ21 and vsv-egfp-sars-cov-2-s aa on three vero cell subtypes and ma104 120 cells demonstrates that the selected variant spreads more efficiently (fig 1b) . screening of a 121 larger panel of cell types (fig 1c) identified ma104 and vero e6 cells as supporting the highest 122 levels of virus production. ectopic expression of tmprss2 led to a further ~10-fold increase in 123 viral titer and larger plaque size (fig 1d) . vsv-sars-cov-2-s δ21 also was capable of infecting 124 calu-3 cells, a human epithelial lung adenocarcinoma cell line (fig s2) . particles but not in the parental vsv (fig 1e) . the protein detected migrated at ~100 133 kilodaltons, a band that corresponds to the cleaved s1 subunit of the glycoprotein (watanabe et 134 al., 2020). to examine whether the s δ21 incorporated into vsv particles is processed to s1 and 135 s2, we performed [ 35 s] cysteine-methionine metabolic labeling in bsrt7 cells, which support 136 robust vsv replication, and analyzed released particles by sds-page and phosphorimaging. in addition to the vsv structural proteins (n, p, m and l), two additional bands were observed 138 for vsv-sars-cov-2-s δ21 that correspond in size to glycosylated s1 (107 kda) and s2 δ21 (85 139 kda) (fig 1f) . negative-stain electron microscopy of sucrose-gradient purified virus particles 140 revealed that the membrane protein projecting from vsv-sars-cov-2-s δ21 is larger than 141 observed on wild-type vsv particles (fig 1g) , which reflects the larger size of the coronavirus 142 spike. we designed a high-throughput assay for titrating sars-cov-2 that could be applied to coronaviruses possess a roughly 30 kb rna genome, which requires that they encode asymptomatic carrier transmission of covid-19 spades: a new genome assembly 591 algorithm and its applications to single-cell sequencing deployment of convalescent plasma 596 for the prevention and treatment of covid-19 vesicular 599 stomatitis virus-based vaccine protects hamsters against lethal challenge with andes virus generation of bovine respiratory 603 syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue 604 culture, and the human rsv leader region acts as a functional brsv genome promoter bbmerge -accurate paired shotgun read merging 608 via overlap ebola virus entry requires the 612 cholesterol transporter niemann-pick c1 endosomal proteolysis of the ebola virus glycoprotein is necessary for infection hiv-1 envelope. effect of the cytoplasmic domain on antigenic 620 characteristics of hiv-1 envelope glycoprotein convalescent plasma as a potential therapy for 623 covid-19 fastp: an ultra-fast all-in-one fastq 626 preprocessor fast algorithms for large-629 scale genome alignment and comparison coronaviruses: an rna proofreading machine regulates replication fidelity and diversity eukaryotic transient-expression 636 system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna 637 polymerase evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of 641 neutralizing antibody responses to sars-cov vesicular stomatitis virus pseudotyped with severe acute 645 respiratory syndrome coronavirus spike protein a single dose of a vesicular stomatitis virus-based 649 influenza vaccine confers rapid protection against h5 viruses from different clades properties of replication-competent vesicular 654 stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses development of a new vaccine for the 659 prevention of lassa fever retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus 663 s protein clinical characteristics of coronavirus disease 2019 in china depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor virus entry. lassa virus entry requires a 676 trigger-induced receptor switch live attenuated recombinant vaccine 680 protects nonhuman primates against ebola and marburg viruses human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute 684 respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells therapeutic strategies in an outbreak scenario to treat the novel coronavirus 687 originating in wuhan, china cov-2 spike pseudotyped virus by recombinant ace2-ig functional assessment of cell entry and receptor 693 usage for sars-cov-2 and other lineage b betacoronaviruses structure of sars coronavirus spike 696 receptor-binding domain complexed with receptor the sequence alignment/map format and samtools a unique strategy for mrna cap methylation used 703 by vesicular stomatitis virus angiotensin-converting enzyme 2 is 707 a functional receptor for the sars coronavirus intracellular targeting signals contribute to 710 localization of coronavirus spike proteins near the virus assembly site the cytoplasmic tail of the severe acute 713 respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval 714 signal that binds copi and promotes interaction with membrane protein synthetic antibodies neutralize sars-cov-2 718 infection of mammalian cells retroviruses pseudotyped with the severe acute respiratory 722 syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting 723 enzyme 2 enhancing dengue virus maturation using a stable furin over-expressing cell line establishment and validation of a pseudovirus neutralization assay for emerging microbes & infections 9 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-735 reactivity with sars-cov cross-neutralization of sars-cov-2 by a human 742 monoclonal sars-cov antibody dipeptidyl peptidase 4 is a functional receptor for the 746 emerging human coronavirus-emc the coronavirus e protein: assembly and beyond. 749 viruses treatment of 5 critically ill patients with covid-19 with convalescent plasma a recombinant vesicular stomatitis virus 756 bearing a lethal mutation in the glycoprotein gene uncovers a second site suppressor that 757 restores fusion specificity, cross-reactivity, and function of 761 antibodies elicited by zika virus infection identification of protective epitopes on ebola virus glycoprotein at the 765 single amino acid level by using recombinant vesicular stomatitis viruses tailored tetravalent antibodies potently and specifically 769 activate wnt/frizzled pathways in cells, organoids and mice human monoclonal antibody 773 combination against sars coronavirus: synergy and coverage of escape mutants structural insights into coronavirus entry an efficient method to make human 780 monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus chimeric viruses with vesicular stomatitis 785 virus: actions in the brain function, and antigenicity of the sars-cov-2 spike glycoprotein site-specific 791 glycan analysis of the sars-cov-2 spike efficient recovery of infectious 794 vesicular stomatitis virus entirely from cdna clones cryo-em structure of the 2019-ncov spike in the prefusion conformation a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-802 sars-cov-2 infection of human small intestinal enterocytes probable pangolin origin of sars-cov-2 associated 809 with the covid-19 outbreak a pneumonia outbreak associated with a new coronavirus of probable bat 813 origin • neutralization can be assessed by bsl2 and bsl3 high-throughput assays • sars-cov-2 and vsv-sars-cov-2-based neutralization assays correlate etoc blurb generate a replication-competent vesicular stomatitis virus (vsv) expressing the sars-cov-2 spike and compare the neutralizing activity of antibodies with vsv-sars-cov-2 to fully infectious sars-cov-2. they show that vsv-sars-cov-2 is a useful bsl2 surrogate virus, as neutralization profiles strongly correlate with focus-reduction neutralization tests using sars-cov-2 key: cord-277823-vijh6x1l authors: teramichi, takurou; fukushi, shuetsu; hachiya, yuma; melaku, simenew keskes; oguma, keisuke; sentsui, hiroshi title: evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of middle east respiratory syndrome coronavirus among livestock in ethiopia date: 2019-11-05 journal: j vet med sci doi: 10.1292/jvms.19-0436 sha: doc_id: 277823 cord_uid: vijh6x1l a serological survey of middle east respiratory syndrome coronavirus (mers-cov) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in ethiopia. the pseudotyped vesicular stomatitis virus coated with the spike protein of mers-cov was used in virus neutralization (vn) tests performed in a biosafety level (bsl)-2 laboratory. the results were similar to those obtained from the vn test using live mers-cov and were more sensitive than the elisa performed using synthetic mers s1 fragment as the antigen as well as the competitive elisa performed using a monoclonal antibody against mers-cov. according to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. moreover, using the present procedure, serological tests for mers-cov can be conducted even in bsl 2 laboratory. pseudovirus vsv-mers/gfp, a recombinant vesicular stomatitis virus pseudotype, integrated with the gfp gene and coated with the spike protein of mers-cov was used in the neutralization test [8] . the infectivity of vsv-mers/gfp was detected based on gfp expression observed using fluorescence microscopy. gfp-expressing cells in photographic images were counted using vh analyzer. the vsv-mers/gfp titers were then expressed as focus forming units (ffu) per ml. cell culture 293t and vero cells were cultivated in dulbecco's modified eagle's medium supplemented with 5% fetal bovine serum (fbs), 100 u/ml of penicillin, and 100 µg/ml of streptomycin at 37°c [8] . 293t cells were used to prepare vsv-mers/gfp and mers-cov recombinant receptor binding domain (rbd) for the celisa antigen. vero cells were used for neutralization tests by live mers-cov and vsv-mers/gfp, respectively. serum samples were collected from dromedary camels (n=38; mean age, 4.3 years; range, 1-13 years), goats (n=25; mean age, 3.9 years; range, 1-8 years), sheep (n=25; mean age, 2.7 years; range, 1-4 years), and cattle (n=15; mean age, 6.7 years; range, 1-11 years) from two herds in bati district, amhara region, and one herd in fafen district, somali region, ethiopia. all animals appeared healthy, shared the same pasturage during the day, and stayed in barns or small grounds specific for each animal species surrounded by fences at night. transportation of the serum samples to japan was conducted with the permission of the japanese government (animal quarantine inspection number nfib070602-011) and followed the rules and regulations of the oie/fao for biological sample transportation. serum from a rabbit immunized with recombinant mers-cov s protein was used as a positive control for neutralization [8] . sera from animals (5 cattle, 5 sheep and 5 goats) reared on the attached farm of nihon university were used as negative controls. the neutralization test using vsv-mers/gfp was performed as previously described [8] . a medium composed of eagle's mem supplemented with 5% fbs was used for virus and serum dilution. serially diluted 0.05 ml of test sera were mixed with equal volumes of 3,000 ffu of vsv-mers/gfp and incubated at 37°c for 1 hr. the mixture was inoculated to vero cells seeded on a 96-well culture plate and incubated at 37°c for 24 hr in a co 2 incubator. gfp-positive cells were then detected using a fluorescence microscope. the number of positive gfp cells was counted as described above. the neutralization titer was determined as the highest serum dilution showing ≤50% of the number of gfp-positive cells compared with the no serum control. neutralization test using live mers-cov was performed as previously described except using vero cells instead of vero-tmprss2 cells [16] . briefly, 0.05 ml of serially diluted test sera was mixed with an equal volume of 100 tcid 50 of mers-cov (emc isolate) in a 96-well culture plate and incubated at 37°c for 1 hr; thereafter,vero cells were added in each well and cultivated at 37°c. cytopathic effects (cpe) on the vero cells were observed at 3 days after infection. the neutralization titer was determined as the highest serum dilution showing at least 50% cpe on the inoculated cells. synthetic s1 fragment of mers-cov was obtained from sino biolobical inc. (beijing, china) and used as the antigen [17] . elisa microplates were coated with 50 µl of 50 ng/ml antigen per well at 4°c overnight, following which the wells were incubated with pbs containing 2% block ace and 0.05% tween 20 for 2 hr at 37°c. following the removal of blocking reagent, diluted serum samples were added and incubated for 1 hr at 37°c. after washing the wells thrice with 0.05% tween 20 in pbs (pbs-t), a peroxidase-labeled protein ag (thermo fisher scientific, waltham, ma, u.s.a.) was added and incubated for 1 hr at 37°c. following further washing thrice with pbs-t, 100 µl of 2,2′-azinobis-3-ethylbenzthiazolinesulfonic acid (abts) substrate solution (roche applied science, penzberg, germany) was added and incubated for 20 min at room temperature. the optical density (o.d.) of each well was measured at 450 nm using a microplate reader, and mean absorbance was determined for each serum sample. one of camel serum that showed a high antibody titer in the neutralization test by live mers-cov was treated as a positive control. the mers-cov rbd was used as the antigen of the celisa. for the preparation of recombinant rbd, the mammalian expression plasmid pcaggs-rbd, which encodes histidine-tagged mers-cov rbd (amino acid 358-588), was transfected to 293t cells. at 2 days after transfection, the recombinant rbd was purified from the supernatant of transfected cells using his-bind purification kit (merck, damastadt, germany). the celisa was performed as described by fukushi et al. [9] . briefly, mers-cov recombinant rbd with pre-determined optimal quantity was coated on a 96-well elisa plate at 4°c for overnight, following which the wells were incubated with pbs containing 2% bovine serum albumin and 0.05% tween 20 (blocking reagent) for 2 hr at 37°c. following the removal of the blocking reagent, 100 µl of a biotin-labeled monoclonal antibody mixed with serially diluted serum samples was added and incubated for 1 hr at 37°c. one of camel serum that showed a high antibody titer in according to the results of the previous study, antibody titers of ≥16 are treated as positive in neutralization test using vsv-mers/gfp. in dromedary camels and cattle, 31 out of 38 and 1 out of 15, respectively, were mers antibody positive. goats and sheep were all mers antibody negative (table l) . moreover, in 15 camels, the antibody titer was 16 or 32; in 7, it was 64 or 128; and in 9 and control positive rabbit serum, it was ≥256. the antibody titer and the positive rate of antibody against mers-cov increased with the age of the camels. the antibody titer in one cow was 64. all sera collected from animals on the attached farm of nihon university were negative. overall, 31 camels and control positive rabbit serum were found to be antibody positive in the neutralization test using mers-cov, and 16 camels were positive in s1-elisa. in the celisa, 26 camels and 1 cow were positive ( table 2 ). the sera collected as negative controls from animals on the attached farm of nihon university were all negative in s1-elisa and celisa. each camel that was antibody positive in s1-elisa or in neutralization tests using mers-cov was found to be positive in neutralization tests using vsv-mers/gfp. cows that were antibody positive in the neutralization test using vsv-mers/gfp or celisa were different animals and both were antibody negative in the neutralization test using mers-cov. a comparison of the antibody titers detected in the neutralization tests using vsv-mers/gfp with those detected using live mers-cov showed a high correlation between both antibody titers, with a correlation coefficient of 0.9753. all camel sera that were positive in any one of the tests-the s1-elisa, celisa, or neutralization tests using live mers-cov-were positive in the neutralization test using vsv-mers/gfp. one cow serum was positive for celisa and another cow was positive in the neutralization test using vsv-mers/gfp, but negative in all other tests. because the neutralization test using live mers-cov is considered the most accurate serological test and because the two positive reactions were observed in only one serological test among the four, these two bovine sera should be considered as nonspecific reactors. the exact reason of nonspecific reaction of these sera remains unclear. the nonspecific reaction may result from differences in immunological conditions of animals or in the degree of hemolysis during the preparation of samples. for example, a nonspecific reaction sometimes appears in cattle after injection with a certain inactivated vaccine in the elisa kit for johne's disease diagnosis in japan. some rhabdovirus cross react with vsv [6] and such a virus might be subclinically infected in ethiopian cattle. there are reports that mers-cov specific antibodies were not detected in sera from cattle, goat and sheep in jordan and saudi arabia, the mers-cov prevalence region [11, 13] . however, they were kept indoor or did not share the pastureland. present results indicate that mers-cov infects only dromedary camels and is unlikely to infect other domestic animals even sharing the pastureland. since the antibody positive rate and antibody titer of mers increased with age, it is considered that mers-cov establishes an infection cycle and inapparently present only among dromedary camels [11] . in present studies, the limited numbers of samples were tested because the import of animal sera from the foot-and-mouth disease endemic region is regulated in japan. further studies using additional samples from other countries would be required to clarify the role of other animal on the ecology and epidemiology of mers-cov. a comparison of the two neutralization tests using vsv-mers/gfp and mers-cov showed a high correlation between both antibody titers. the former showed an antibody titer of ≥256, whereas the latter showed an antibody titer of ≥512. s1-elisa was not sensitive compared to other tests because only 16 serum samples were positive and they required an antibody titer of ≥64 in vsv-mers/gfp. however, the sensitivity of s1-elisa would be improved by examining the cut-off value and dilution of the test serum. in celisa, five serum samples were negative out of the 31 positive samples in the neutralization tests, and an antibody titer of 16 was observed in both neutralization tests. therefore, neutralization test using vsv-mers/gfp exhibits sensitivity similar to the neutralization test using mers-cov and is more sensitive compared with the s1-elisa as well as celisa. the present study shows that the neutralization test using vsv-mers/gfp, s1-elisa, and celisa are as specific to mers-cov infection as the serological tests, although their sensitivities slightly differ. the detection of antibody and epidemiological survey of mers-cov could be possible by appropriately selecting these tests according to the desired purpose, without using a bsl-3 laboratory. middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates ksa mers-cov investigation team 2013. hospital outbreak of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group cross-neutralization between vesicular stomatitis virus type indiana and chandipura virus transmission of mers-coronavirus in household contacts inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein characterization of novel monoclonal antibodies against the mers-coronavirus spike protein and their application in species-independent antibody detection by competitive elisa middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus infection not found in camels in japan characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china acute middle east respiratory syndrome coronavirus infection in livestock dromedaries isolation of a novel coronavirus from a man with pneumonia in saudi arabia acknowledgment. this work was partly supported by jsps kakenhi (grant number 17h04642), japan agency for medical research and development (grant number jp18fk0108058) and the academic frontier project for private universities (s1491007) from the ministry of education, culture, sports, science and technology of japan. key: cord-326013-5i35zdmv authors: carpinteiro, alexander; edwards, michael j.; hoffmann, markus; kochs, georg; gripp, barbara; weigang, sebastian; adams, constantin; carpinteiro, elisa; gulbins, anne; keitsch, simone; sehl, carolin; soddemann, matthias; wilker, barbara; kamler, markus; bertsch, thomas; lang, karl s.; patel, sameer; wilson, gregory c.; walter, silke; hengel, hartmut; pöhlmann, stefan; lang, philipp; kornhuber, johannes; becker, katrin anne; ahmad, syed a.; fassbender, klaus; gulbins, erich title: pharmacological inhibition of acid sphingomyelinase prevents uptake of sars-cov-2 by epithelial cells date: 2020-10-29 journal: cell rep med doi: 10.1016/j.xcrm.2020.100142 sha: doc_id: 326013 cord_uid: 5i35zdmv the acid sphingomyelinase/ceramide system plays an important role in bacterial and viral infections. here we report that either pharmacological inhibition of acid sphingomyelinase with amitriptyline, imipramine, fluoxetine, sertraline, escitalopram, or maprotiline, or genetic downregulation of the enzyme prevents infection of cultured cells or freshy isolated human nasal epithelial cells with sars-cov-2 or pseudoviral pp-vsv-sars-cov-2 particles expressing spike, a bona fide system mimicking sars-cov-2 infection. infection activates acid sphingomyelinase and triggers a release of ceramide on the cell surface. neutralization or consumption of surface ceramide reduces infection with pp-vsv-sars-cov-2 spike. treating volunteers with a low dose of amitriptyline prevents infection of freshly isolated nasal epithelial cells with pp-vsv-sars-cov-2 spike. the data justify clinical studies investigating whether amitriptyline, a safe drug used clinically for almost 60 years, or other antidepressants that functionally block acid sphingomyelinase prevent sars-cov-2 infection. infections with a novel member of the coronaviridae family were reported in late 2019 in wuhan, china 1 . the virus was named severe acute respiratory syndrome coronavirus-2 (sars-cov-2). subsequently, the virus spread globally and is responsible for the coronavirus disease 2019 (covid19) pandemic. infection with sars-cov-2 often results in mild respiratory tract disease, but a substantial number of patients also experience severe symptoms and pneumonia, and ∼70% of these critically ill patients require intensive care and ventilator treatment, with a mortality rate of 62% 2 . even when the large number of only mildly affected patients are included, the mortality rates are higher than those associated with seasonal influenza 3,4 . cellular infection with sars-cov-2 is initiated by the binding of the surface unit s1 of the viral spike glycoprotein to its cellular receptor angiotensin-converting enzyme 2 (ace2), resulting in cleavage of the viral spike protein by the activity of transmembrane serine protease 2 (tmprss2) or cathepsin l, and in viral entry [5] [6] [7] [8] . although the binding of the virus to its receptor has been elucidated in detail [6] [7] [8] , the changes that occur in the host cell membrane during viral processing and entry require definition. membrane changes that mediate viral entry may be a very promising target for preventing the infection. previous studies have used replication-deficient vesicular stomatitis virus (vsv) pseudoviral particles (pp-vsv) presenting coronavirus spike protein (pp-vsv-sars-cov-2 spike) on their surface. studies have shown that these particles accurately reflect key aspects of the entry of coronavirus into host cells 5 . these particles were previously shown to bind to ace2 for infectious entry, and entry was inhibited by anti-ace2 antibodies 5 . thus, these particles are a bona fide model for studying the events of sars-cov-2 entry. we have previously shown that acid sphingomyelinase and ceramide play an important role in receptor signaling and infection biology 9, 10 . acid sphingomyelinase (ec 3.1.4.12, sphingomyelin phosphodiesterase; optimal ph 5.0) is a glycoprotein that functions as a lysosomal hydrolase, catalyzing the degradation of sphingomyelin to phosphorylcholine and ceramide. acid sphingomyelinase is present in lysosomes, j o u r n a l p r e -p r o o f 5 but because these compartments are constantly recycling to the plasma membrane it can also be found on the cell surface 9, 10 . the activity of acid sphingomyelinase on the cell surface results in the formation of ceramide in the outer leaflet of the cell membrane. the generation of ceramide molecules within the outer leaflet alters the biophysical properties of the plasma membrane, because the very hydrophobic ceramide molecules spontaneously associate with each other to form small ceramide-enriched membrane domains that fuse and form large, highly hydrophobic, tightly packed, gel-like ceramide-enriched membrane domains [10] [11] [12] [13] . in addition, ceramide has been shown to directly bind to a variety of proteins, including cathepsin d 14 , phospholipase a 2 15 , ceramide-activated protein serine/threonine phosphatases (capp) 16 , protein kinase c isoforms 17, 18 , and lc3b-ii 19 . many antidepressants functionally inhibit acid sphingomyelinase activity [20] [21] [22] [23] [24] [25] . these cationic amphiphilic drugs indirectly inhibit acid sphingomyelinase activity by displacing the enzyme from lysosomal membranes, in particular intralysosomal vesicles, thereby releasing the enzyme into the lysosomal lumen and causing its partial degradation [20] [21] [22] [23] [24] [25] . we have previously shown that rhinovirus infections activate acid sphingomyelinase and lead to the formation of ceramide and ceramide-enriched membrane domains. amitriptyline, sertraline, and other functional inhibitors of acid sphingomyelinase activity (fiasmas) inhibit cellular infection with rhinovirus 26 . similar observations have been made regarding infections with ebola virus 27 demonstrating that amitriptyline and other fiasmas inhibit infection with ebola virus in vitro 27 . because some antidepressants are widely used in clinical practice and have a very favorable safety profile, we investigated whether these drugs could be repurposed to treat or prevent infections with sars-cov-2. our results show that acid sphingomyelinase is activated within 20 to 30 minutes to determine whether acid sphingomyelinase can be targeted to prevent the infection of cells with sars-cov-2, we treated vero cells with the fiasma amitriptyline [22] [23] [24] or left the cells untreated. we then exposed the cells to pp-vsv-sars-cov-2 spike next, we determined whether amitriptyline also affects the infection of human caco-2 cells with authentic sars-cov-2. amitriptyline-pretreated caco-2 cells were infected with sars-cov-2, and the replication of sars-cov-2 was monitored by reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) using two separate primer sets and a plaque assay. the results showed that amitriptyline also reduced the infection of human cells with sars-cov-2 by approximately 90% (fig. 2 ). j o u r n a l p r e -p r o o f 8 the results of these studies suggested that acid sphingomyelinase serves an important function in sars-cov-2 infection. to confirm the role of acid sphingomyelinase in infection with sars-cov-2 independent of pharmacological inhibition, we used shrna-mediated suppression of the expression of acid sphingomyelinase in caco-2 cells. we confirmed the downregulation of acid sphingomyelinase by measuring the activity of the enzyme in lysates of uninfected cells (fig. 3a) . the results showed that genetic downregulation of acid sphingomyelinase prevented infection with pp-vsv-sars-cov-2 spike (fig. 3a ). control shrna constructs had no effect on cellular infection with pp-vsv-sars-cov-2 spike. we further tested whether neutral sphingomyelinase may also be involved in cellular infection with sars-cov-2. to this end, we transfected caco-2 cells with shrna targeting neutral sphingomyelinase 2 and control shrna. transfection reduced neutral sphingomyelinase activity by approximately 60% but had no effect on cellular infection with pp-vsv-sars-cov-2 spike (fig. 3a) . we next investigated whether infection with pp-vsv-sars-cov-2 spike activates the acid sphingomyelinase/ceramide system. the results showed that treating vero cells with pp-vsv-sars-cov-2 spike resulted in rapid activation of acid sphingomyelinase and a concomitant release of ceramide (fig. 3b, fig. 4a ). in contrast, treating vero cells with pp-vsv lacking sars-cov-2 spike or with pp-vsv-g particles did not result in the activation of acid sphingomyelinase or in a release of ceramide (fig. 3b, fig. 4a ), a finding indicating that the activation of acid sphingomyelinase and the release of ceramide are specifically induced by the spike protein. pretreatment of the cells with 5, 10, 20, or 25 µm amitriptyline prevented the activation of acid sphingomyelinase and the release of ceramide upon infection with pp-vsv-sars-cov-2 spike for 30 min (fig. 3b, fig. 4a ). controls showed that amitriptyline reduced both the constitutive and the viral-induced activity of acid sphingomyelinase (fig. 3b ). treating vero cells with neutralizing antibodies to spike or with recombinant ace2 protein prevented the activation of acid sphingomyelinase and the release of ceramide upon infection with pp-vsv-sars-cov-2 spike (fig. 3b, fig. 4a amitriptyline and other drugs with similar structure and properties have been clinically used for many years (since 1962) to treat patients with depressive disorder. in the volunteer studies we used a low dose of amitriptyline, i.e., 0.5 mg/kg, which is lower than the dose usually used to treat patients with major depression (75-150 mg/day, i.e., approximately 1-2 mg/kg). at this dose, the drug has only mild effects, such as dry mucosa and tiredness. however, at higher dosages (higher than 100 mg/day, twice the dose we used or higher), amitriptyline and many other antidepressants may induce a cardiac qt elongation as a possible (but rare) adverse effect. qt elongation can result in cardiac arrhythmia and serious cardiac adverse effects 29 . thus, it may be advisable to perform an electrocardiogram (ecg) before the first administration of amitriptyline or any of the other fiasmas used here to exclude a pre-existing qt elongation. amitriptyline and other antidepressants acting as fiasmas exert no known adverse effects on the immune system. our studies demonstrate that even low dosages of amitriptyline provide long-lasting and very efficient protection against infection. our findings also indicate that other drugs, such as imipramine, desipramine, fluoxetine, sertraline, escitalopram, and maprotiline, with concentrations similar to that of amitriptyline 22 our studies are strongly supported by a recently pre-printed study 30 that showed a marked beneficial effect of antidepressants on the clinical course of covid-19. hospitalized patients receiving antidepressants had a much better outcome, determined as decreased medical necessity of intubation or death, than patients receiving no antidepressants. best results were obtained with venlafaxin, fluoxetine, escitalopram and mirtazipine, drugs that were also shown in the present study to inhibit acid sphingomyelinase and ceramide release upon pp-vsv-sars-cov-2 spike infection. it is important to note that the dose of antidepressants used in this j o u r n a l p r e -p r o o f 13 retrospective study was not optimized. amitriptyline was used at a medium dose of 26 mg/day, which is a rather low dose. amitriptyline is often used at these low dosages for sedation and not as an antidepressant, while other drugs such as fluoxetine and escitalopram are always used as antidepressants and applied at higher dosages. further, fluoxetine also inhibits sars-cov-2 replication 31 , an effect that was not investigated in the present study, since the pseudoviral particles do not replicate. amitriptyline and many other antidepressants are weak bases that are protonated in lysosomes and thereby trapped in lysosomes, but they also localize in acidic subcompartments of the cell membrane. because of their physicochemical properties (for instance for amitriptyline: high lipophilicity and weak basicity; logp = 4.92, pka = 9.4, www.drugbank.ca), these drugs accumulate in acidic intracellular compartments 32 . this accumulation leads to a high tissue concentration and accordingly to a high volume of distribution (for instance for amitriptyline: vd = 16 l/kg, www.drugbank.ca). thus, high tissue concentrations of these drugs can be detected in all organs. in fact, the highest uptake of amitriptyline among all tissues with a lung/blood concentration gradient of approximately 50 is found in the lungs of mice and rats 33, 34 . in humans, even higher concentration gradients have been found between lung and blood 35 . because of the particularly high accumulation in the lung, the antiviral in vitro concentrations (up to 25 µm) that we found to be effective are likely to be achieved with conventional oral therapy of amitriptyline. we also expect a considerably longer elimination half-life of amitriptyline in lung tissue than in blood because of the high concentration of these drugs in deep compartments, such as lysosomes. the organic ring system of amitriptyline and other fiasmas may bind to the lipid membrane, whereas the protonated tertiary amine displaces acid sphingomyelinase from lysosomal membranes or from the plasma membrane within acidic subdomains. thus, these weak bases do not directly inhibit acid sphingomyelinase activity but rather functionally inhibit it. this mode of action also explains why antidepressants do not completely inhibit acid sphingomyelinase activity and do not induce severe adverse effects in clinical use. in addition, our studies indicate that the administration of anti-ceramide antibodies or neutral ceramidase also protects against sars-cov-2 infections. anti-ceramide j o u r n a l p r e -p r o o f 14 antibodies and neutral ceramidase are not approved for clinical use, but could be easily tested for systemic or local adverse effects before a potential treatment of sars-cov-2 infections. furthermore, both anti-ceramide antibodies and neutral ceramidase could potentially be administered via inhalation or as a nasal spray to prevent sars-cov-2 infections. the molecular mechanisms how ceramide mediates infection of epithelial cells with sars-cov-2 remain to be determined. ceramide has been shown to directly activate several enzymes and to form large, highly hydrophobic ceramide-enriched membrane domains that serve to re-organize receptor and signalling molecules [10] [11] [12] [13] . it remains to be determined whether sars-cov-2 also induces these platforms and whether ceramide and/or these platforms are required for internalization or activation of tmprss2 and cathepsin l that mediate processing of spike require for fusion with the cell membrane 36 . high ceramide levels have been linked with hypertension and obesity 37, 38 the authors declare no competing financial interests. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, erich gulbins (erich.gulbins@uni-due.de). this study did not generate new unique reagents. the published article includes all datasets generated or analyzed during the study. human nasal epithelial cells were obtained from 6 healthy volunteers, 4 women and 2 men. their ages were 20, 21, 43, 48, 54, and 56. we did not observe any differences in infection between cells from women or men. sample size was planned for the continuous variable difference in viral uptake and was based on two-sided we obtained nasal epithelial cells from these volunteers immediately before oral administration of 0.5 mg/kg amitriptyline (neuraxpharm, germany) and again 1. caco-2 colon epithelial cells (atcc htb-37) were cultured in dmem supplemented as described above. pseudotyped viral particles based on a replication-deficient vsv that codes for egfp and firefly luciferase instead of parental vsv-g (vsv*∆g-fluc) 39 a further aliquot of the nasal epithelial cells was immediately shock frozen in liquid nitrogen after removal for determination of acid sphingomyelinase activity (see below). nasal epithelial cells were also obtained from untreated volunteers as described above and were treated with 10 µm amitriptyline (sigma; # a 8404) dissolved in 0.9% nacl, anti-ceramide or control antibodies, neutral ceramidase (see below) or left untreated for 60 min ex vivo and then infected with pp-vsv-sars-cov-2 spike particles for 60 min. cells were then washed and further incubated for 24 h to allow expression of egfp. infection was analyzed as above. vero cells were grown on glass coverslips, treated with 10 or 25 µm amitriptyline as ceramide amounts were determined by comparison with a standard curve using c16 to c24 ceramides as substrates. to investigators were blinded to the identity of the samples in all microscopy experiments. all data are available upon request from the authors. authors confirm that all data are included in the manuscript. a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural and functional basis of sars-cov-2 entry by using human ace2 cd95 signaling via ceramide-rich membrane rafts host defense against pseudomonas aeruginosa requires ceramide-rich membrane rafts raft ceramide in molecular medicine compartmentalization of ceramide signaling: physical foundations and biological effects observation of topical catalysis by sphingomyelinase coupled to microspheres cathepsin d targeted by acid sphingomyelinase-derived ceramide ceramide binds to the calb domain of cytosolic phospholipase a2 and facilitates its membrane docking and arachidonic acid release ceramide-activated protein phosphatase: partial purification and relationship to protein phosphatase 2a pkc zeta is a molecular switch in signal transduction of tnf-alpha, bifunctionally regulated by ceramide and arachidonic acid selective ceramide binding to protein kinase c-alpha and -delta isoenzymes in renal mesangial cells targeting flt3-itd signaling mediates ceramide-dependent mitophagy and attenuates drug resistance in aml the tricyclic antidepressant desipramine causes proteolytic degradation of lysosomal sphingomyelinase in human fibroblasts interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model acid sphingomyelinase/ceramide system mediates effects of antidepressant drugs antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide emerging mechanisms of drug-induced phospholipidosis rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection sars-cov-2 receptor ace2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increase of heart rate and qtc by amitripytline, but not by venlafaxine, is correleated to serum concentration association between ssri antidepressant use and reduced risk of intubation or death in hospitalized patients with coronavirus disease 2019: a multicenter retrospective observational study the serotonin reuptake inhibitor fluoxetine inhibits sars-cov-2 quantitative modeling of selective lysosomal targeting for drug design distribution and fate of c14-amitriptyline in mice and rats postmortem release of amitriptyline from the lungs; a mechanism of postmortem drug redistribution postmortem distribution of tramadol, amitriptyline, and their metabolites in a suicidal overdose efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 ceramide is upregulated and associated with mortality in patients with chronic heart failure the unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multispecies type i interferon mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization key: cord-262752-bwofzbwa authors: li, qianqian; liu, qiang; huang, weijin; li, xuguang; wang, youchun title: current status on the development of pseudoviruses for enveloped viruses date: 2017-12-07 journal: rev med virol doi: 10.1002/rmv.1963 sha: doc_id: 262752 cord_uid: bwofzbwa emerging and reemerging infectious diseases have a strong negative impact on public health. however, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. as pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. however, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. this review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields. a pseudovirus is a recombinant viral particle with its core/backbone and envelope proteins derived from different viruses 1 ; moreover, the genes inside the pseudovirus are usually altered or modified so that they are unable to produce the surface protein on their own. as such, an additional plasmid or stable cell line expressing the surface proteins is needed to make the pseudovirus. 2 pseudoviruses are capable of infecting susceptible cells, but they only replicate for 1 round in the infected host cells. 3 compared with wild-type (wt) viruses, pseudoviruses can be safely handled in biosafety level (bsl)-2 laboratories 4 and are usually easier to manipulate experimentally. 5 nevertheless, the conformational structure of pseudoviral surface proteins bears high similarity to that of the native viral proteins, and these surface proteins can effectively mediate viral entry into host cells. therefore, pseudoviruses are widely used for the study of cellular tropism, 6 receptor recognition, 7 and virus inhibition, 8 as well as for developing and evaluating antibodies 9 and vaccines. 10 in addition, data from in vitro pseudovirus-based antiviral assays and in vivo biodistribution analyses have been found to correlate very well with the results generated by using live wt viruses. 11, 12 as pseudoviruses have usually been engineered to carry reporter genes, it is much easier to perform quantitative analyses on these viruses than on wt viruses, 13 and the number of pseudovirus-infected cells has been shown to be directly proportional to reporter gene expression. the reporter genes usually encode either an enzyme or a fluorescent protein, with each option having its particular strengths and weaknesses. specifically, chemiluminescence assays usually have lower background and are more sensitive, but the data acquisition and analyses for these assays are time-consuming and more expensive. in contrast, assays using a florescence protein, such as green fluorescent protein, are cheaper and easier to operate in both in vitro and in vivo systems; however, they are less sensitive and may have higher background. [14] [15] [16] in this review, we provide an update on the development and application of pseudoviral systems and discuss some challenging technical issues. the hiv-1 packaging system is the most widely used pseudovirus packaging system. to make this packaging system, hiv genes are selectively cloned into dna vectors. specifically, 2 to 4 plasmids are used as the vectors, a strategy that aims to minimize viral gene recombination and thereby reduce the possibility of reversion to the wt virus. table 1 lists the currently used hiv-1-based systems. the original 2-plasmid system comprises 1 envelope plasmid and 1 hiv-1 backbone plasmid, ie, psg3δenv and pnl4-3 (the env gene sequence in psg3δenv is destroyed). 51 however, this system is not ideal, as its viral yield is usually very low. improvements have been made through the addition of other sequences for better reporter gene expression. specifically, our research group inserted the reporting gene, firefly luciferase (fluc), into psg3δenv between env and nef to produce psg3δenv.fluc.δnef. in addition, we also generated psg3δenv.cmvfluc, which carries a functional nef and cmv promoter to drive the reporter gene. 10 by using these optimized backbone and envelope protein expression plasmids, our group succeeded in producing several hiv pseudoviruses carrying the envelope proteins of ebov, marv, lasv, middle east respiratory syndrome-coronavirus, rabies virus (rv), chikungunya virus, and nipah virus (niv). the yields of pseudoviruses constructed with this optimized system were improved by 100 to 1000-folds as compared with those of pseudoviruses constructed with pnl4-3.luc.r-e. 52 the hiv 3-plasmid system is usually comprised of a packaging plasmid, a transfer plasmid containing the reporter gene, and an envelope-expressing plasmid. specifically, this system is made by splitting the hiv-1 backbone into separate packing and transfer plasmids. the packaging plasmid expresses the gag and pol proteins, while the transfer plasmid contains the cis-regulatory elements needed for hiv reverse transcription, integration, and packaging as well as multiple cloning sites and a reporter gene under the control of a heterogeneous promoter. 48-50 the envelope-expressing plasmid is made of a vector carrying the envelope gene driven by a cmv promoter. the hiv 4-plasmid system is based on the 3-plasmid system, with the rev protein being expressed by an additional, separate plasmid. specifically, this system comprises 1 packaging plasmid expressing the gag and pol proteins, a second packaging plasmid encoding rev, 1 plasmid producing the wt envelope protein, and a transfer plasmid with cis-regulatory elements. 53 these 3 hiv-based systems were reported by different groups, and, as yet, no comparison has been made among the different systems in safety and efficiency. our group is able to drastically improve the viral yield with 2-plasmid system, while no safety issue of this hiv pseudoviral systems was observed in animals. 52 2.1.2 | the simian immunodeficiency virus packaging system hiv env cellular tropism, neutralization antibody assay, impact of env amino acid mutation, and glycosylation on the neutralization epitope, drug screening and validation, and receptor recognition psg3δenv; pnl4-3.luc.r-e the vsv packaging system is a versatile tool for making pseudotyped viruses; this system is advantageous in that it has no stringent selectivity for the envelope proteins, and the resulting virus may be manipulated in a bsl-2 laboratory. early studies jointly employed vsv and a second virus to coinfect cells, resulting in the pseudotyped virus carrying the core of vsv with envelope proteins derived from the other virus. 60 stillman et al were the first to clone the vsv genome into a plasmid to make stable vsv, 61 which was subsequently used to generate pseudoviruses carrying heterogeneous glycoprotein. 62, 63 various reporter genes were successively inserted into this plasmid to facilitate its easy detection. 64, 65 some examples of vsv-based pseudovirus system are listed in table 2 . notably, when the vsv packaging system is used to make a pseudovirus, there may be residual vsv virus mixed with the pseudovirus, thereby complicating the neutralization assay in which it is used or producing false-positive results. preferably, the amount of vsv should be minimized; however, if excess vsv is believed to be interfering with a pseudovirus-based assay, treatment of the pseudovirus preparation with a vsv neutralizing antibody could be considered before its use in future assays. the mlv packaging system, also called the retroviral system, is commonly used to make pseudoviruses. table 3 lists the pseudoviruses packaged by the mlv system that have been reported in the literature. early work by witte and colleagues showed that when they used vsv to infect the cells in which mlv is packaged, they were able to harvest pseudovirus for use in neutralization antibody assays. 98 since then, the genome of mlv had been split into 2 parts: one encoding gag-pol and the other containing the reporter gene. the 2 gene sets were further cloned into plasmids to generate highly efficient mlv packaging systems. 99 to improve the stability of this system, investigators established several cell lines that were confirmed to be stable in transfection and expression. murine leukemia virus may actually be a better choice than hiv as a packaging system in some cases. for example, in studying lasv-mediated entry into cells, cosset et al compared the mlv and hiv systems and found that the former is 5-fold more efficient than the latter. 100 the aforementioned pseudovirus packaging systems have not always been successful in generating certain types of pseudoviruses. in those cases, other alternatives such as reverse genetics have been reported. for example, hu et al prepared a pseudotyped dengue virus (denv) types 1 to 4 by using the hiv system, but its titer was insufficiently high. 101 however, by using reverse genetics, reporter genes were a high yield is needed for practical applications of the pseudoviruses. there are several factors that can critically influence their yield/titer. in general, the subcellular localization for viral packaging and maturasuccessfully used the mlv packaging system to generate a pseudotyped influenza virus. 108 cosset et al reached the same conclusion for pseudotyped lasv preparations. 100 moreover, in our experiences, pseudotyped lasv packaged by the vsv system had a higher titer than that produced by using the hiv system. furthermore, the vsv system was able to incorporate hantavirus glycoprotein that had failed to be packaged by hiv. nonetheless, our group developed a modification of the hiv packaging system that could improve the pseudoviral yield by 100-fold; specifically, the backbone plasmid psg3δenv.cmvfluc that was developed in our lab was superior to pnl4-3.luc.r-e-. 10 the packaging conditions can also drastically influence the pseudovirus yield. in the hiv packaging system, the pseudovirus titer could be pseudoviruses have been widely used for conducting in vitro studies on the interaction between the virus and the host cells. 112 they have also proven to be very useful for in vivo studies, particularly studies on the mechanism of viral infection as well as on the biodistribution. 113 our lab used a pseudotyped rv carrying reporter genes to establish an in vivo imaging model in mice. this mouse model was used to study viral tissue tropism and its dynamic change over time. 10 we also established a pseudotyped ebov mouse model; the ebov pseudoviruses were mainly detected in the thymus and spleen following viral infection, revealing that the pseudotyped ebov and wt ebov have the same tissue tropism. 52 other groups have also used pseudotyped hsv-1 and marv in small animal models to investigate viral infections. 114 4.2 | application of pseudoviral systems to neutralization antibody and antibody-dependent cellmediated cytotoxicity assay antibody neutralization assay based on pseudoviruses has been widely used, particularly for the analyses of some virulent viruses that would otherwise need to be handled in bsl-3 or bsl-4 laboratories. compared with the traditional assays, the reported pseudovirus-based assays have demonstrated a good correlation with the wt virus-based assay; the pseudovirus-based assays are usually high-throughput procedures with fewer amounts of serum samples needed. 1, 104, 115, 116 wilkinson et al compared 22 platform technologies for assaying antibody against ebov with neutralization assays by using the wt virus and found that the 5 best assays included methods based on wt and vsv pseudotype neutralization and elisa, but the lentiviral and other platforms were problematic. 117 no false start for novel pseudotyped vectors vesicular stomatitis virus pseudotypes of retroviruses a bioluminescent imaging mouse model for marburg virus based on a pseudovirus system lassa and ebola virus inhibitors identified using minigenome and recombinant virus reporter systems high-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses infectious hepatitis c virus pseudo-particles containing functional e1-e2 envelope protein complexes transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses a comparative high-throughput screening protocol to identify entry inhibitors of enveloped viruses most neutralizing human monoclonal antibodies target novel epitopes requiring both lassa virus glycoprotein subunits development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison a safe and sensitive enterovirus a71 infection model based on human scarb2 knock-in mice comparison of two high-throughput assays for quantification of adenovirus type 5 neutralizing antibodies in a population of donors in china development of a moloney murine leukemia virus-based pseudotype anti-hiv assay suitable for accurate and rapid evaluation of hiv entry inhibitors characterization of retroviral and lentiviral vectors pseudotyped with xenotropic murine leukemia virus-related virus envelope glycoprotein development of a triple-color pseudovirion-based assay to detect neutralizing antibodies against human papillomavirus temperature-dependent production of pseudoinfectious dengue reporter virus particles by complementation dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e1 glycoprotein development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection chikungunya virus glycoproteins pseudotype with lentiviral vectors and reveal a broad spectrum of cellular tropism filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo toremifene interacts with and destabilizes the ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses in vitro evaluation of cyanovirin-n antiviral activity, by use of lentiviral vectors pseudotyped with filovirus envelope glycoproteins packaging hiv-or fiv-based lentivector expression constructs and transduction of vsv-g pseudotyped viral particles pseudotype formation between enveloped rna and dna viruses replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines quantification of lyssavirus-neutralizing antibodies using vesicular stomatitis virus pseudotype particles second generation of pseudotype-based serum neutralization assay for nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase a system for functional analysis of ebola virus glycoprotein rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors characterization of pseudotype vsv possessing hcv envelope proteins use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test a pseudotype vesicular stomatitis virus containing hantaan virus envelope glycoproteins g1 and g2 as an alternative to hantavirus vaccine in mice study of andes virus entry and neutralization using a pseudovirion system efficient production of hantaan and puumala pseudovirions for viral tropism and neutralization studies analyses of entry mechanisms of novel emerging viruses using pseudotype vsv system characterization of the interaction of lassa fever virus with its cellular receptor alpha-dystroglycan analysis of lujo virus cell entry using pseudotype vesicular stomatitis virus development of a neutralization assay for nipah virus using pseudotype particles ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus a neutralization test for specific detection of nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein involvement of ceramide in the propagation of japanese encephalitis virus preparation of vesicular stomatitis virus pseudotype with chikungunya virus envelope protein efficient generation of vesicular stomatitis virus (vsv)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses a vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza a virus analysis of the entry mechanism of crimean-congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines murine leukemia virus pseudotypes of la crosse and hantaan bunyaviruses: a system for analysis of cell tropism truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles assessment of hiv-1 entry inhibitors by mlv/hiv-1 pseudotyped vectors functional murine leukemia virus vectors pseudotyped with the visna virus envelope show expanded visna virus cell tropism ross river virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production coreceptor switch of [mlv(sivagm)] pseudotype vectors by v3-loop exchange development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein murine leukemia virus (mlv)-based coronavirus spike-pseudotyped particle production and infection transduction of schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped moloney murine leukemia retrovirus transduction of schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies detection of antibodies against h5 and h7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus a transient three-plasmid expression system for the production of high titer retroviral vectors characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles characterization of retrovirusbased reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses a rapid and quantitative assay for measuring antibody-mediated neutralization of west nile virus infection a high-throughput assay using dengue-1 virus-like particles for drug discovery pseudotypes: your flexible friends a plasma membrane localization signal in the hiv-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into vsv virions truncation of the enzootic nasal tumor virus envelope protein cytoplasmic tail increases env-mediated fusion and infectivity polybasic kkr motif in the cytoplasmic tail of nipah virus fusion protein modulates membrane fusion by inside-out signaling a sensitive retroviral pseudotype assay for influenza h5n1-neutralizing antibodies optimized large-scale production of high titer lentivirus vector pseudotypes a high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes the optimisation of pseudotyped viruses for the characterisation of immune responses to equine influenza virus pseudotyping viral vectors with emerging virus envelope proteins applications of bioluminescence imaging to the study of infectious diseases transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination optimization and proficiency testing of a pseudovirus-based assay for detection of hiv-1 neutralizing antibody in china comparison of platform technologies for assaying antibody to ebola virus novel cross-reactive monoclonal antibodies against ebola virus glycoproteins show protection in a murine challenge model high-throughput screening of viral entry inhibitors using pseudotyped virus teicoplanin inhibits ebola pseudovirus infection in cell culture identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay a systematic screen of fdaapproved drugs for inhibitors of biological threat agents characterization of the inhibitory effect of an extract of prunella vulgaris on ebola virus glycoprotein (gp)-mediated virus entry and infection properties of wild-type, c-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral vector particles cholesterol supplementation during production increases the infectivity of retroviral and lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (vsv-g) short communication: potential risk of replication-competent virus in hiv-1 env-pseudotyped virus preparations current status on the development of pseudoviruses for enveloped viruses the authors have no competing interest. key: cord-290243-m8yfugr0 authors: kim, kyung ran; moon, hyung ryong; park, ah-young; chun, moon woo; jeong, lak shin title: design, synthesis, and biological evaluation of novel iso-d-2′,3′-dideoxy-3′-fluorothianucleoside derivatives date: 2007-01-01 journal: bioorganic & medicinal chemistry doi: 10.1016/j.bmc.2006.09.066 sha: doc_id: 290243 cord_uid: m8yfugr0 abstract novel iso-d-2′,3′-dideoxythianucleoside derivatives 1–4 were designed and asymmetrically synthesized as a bioisostere of lamivudine to search for new anti-hiv agents. the information about using sulfur participation occurred on dast fluorination and mitsunobu reaction will be of great help in synthesizing sulfur-containing compounds. final compounds 1–4 were evaluated against hiv-1 and 2, hsv-1 and 2, emcv, cox. b3, vsv, flua (taiwan), flua (johan.), fcv, and fip. only cytosine analogue 3 showed a potent anti-vsv activity (ec50 =9.43μg/ml). this result implies that iso-2′,3′-dideoxy sugar templates might play a role of a sugar surrogate of nucleosides for the development of anti-rna virus agent. since zidovudine (azt) 1 has been known as a potent anti-human immunodeficiency virus (anti-hiv) agent, a number of nucleoside analogues have been designed, synthesized, and their antiviral activities were evaluated against a variety of viruses including hiv for the development of antiviral agents. particularly, 2 0 ,3 0 -dideoxynucleosides (ddns) have exhibited potent anti-hiv activity. among them, didanosine (ddi), 2 zalcitabine (ddc), 3 and stavudine (d4t) 4 have been launched as drugs for the treatment of aids patients (fig. 1) . however, these 2 0 ,3 0 -ddns have several drawbacks such as easy cleavage of their glycosidic bond under acidic conditions similar to a gastric environment and catabolism by adenosine deaminase (ada), adenosine-5 0 -monophosphate (amp) deaminase, and purine nucleoside phosphorylase (pnp). also, lamivudine (3tc) 5 has been clinically used for the treatment of aids and hbv (hepatitis b virus) infec-tion (fig. 2) . structural characteristics of lamivudine are that there is no hydroxyl substituent at both its 2 0 -and 3 0 -positions like 2 0 ,3 0 -ddn analogues and are two heteroatoms on the sugar moiety. in addition, lamivudine belongs to l l-nucleoside unlike other anti-aids drugs. isonucleosides 6 in which their base is moved into c2 0 -position from anomeric position have been synthesized and evaluated for anti-hiv activity. among them, iso-dda and iso-ddg were found to be active against hiv-1 without cytotoxicity and this class of nucleosides were known to possess the intrinsic metabolic advantages such as resistance to ada and enhanced stability of glycosidic bond under acidic and enzymatic conditions compared to natural nucleosides and 2 0 ,3 0 -ddns including lamivudine. 6a,7 on the other hand, a fluorine atom has been used as a good bioisostere because many fluorinated nucleosides such as 2 0 -f-dda, 8 2 0 -f-ara-ddc, 9 and flt 10 were found to exhibit significant antiviral activities. furthermore, it is worthy to note that l l-nucleoside analogues such as lamivudine can be considered as iso-d d-nucleoside analogue as shown in figure 2 . therefore, as a part of our continuous efforts searching for novel antiviral agents, it was interesting to design and synthesize iso-d d-2 0 ,3 0 -dideoxythianucleoside derivatives with a 3 0 -fluoro substituent and to evaluate them against various viruses because ch-f group might act as a bioisostere of sugar ring oxygen of lamivudine and/or as a hydrogen bonding acceptor at the active site of their target enzyme, and because the d d-sugar skeleton of iso-d d-2 0 ,3 0 -dideoxythianucleosides resembles l l-1,3oxathiolane ring of lamivudine. here, we wish to report the asymmetric synthesis and biological activities of novel fluorinated iso-d d-2 0 ,3 0 -dideoxythianucleosides 1-4. a strategy for the synthesis of glycone of iso-d d-2 0 ,3 0dideoxy-3 0 -fluorothianucleosides is outlined in scheme 1. it was envisioned that 1,4-anhydro-4-thioarabitol 6 derived from d d-glucose via 1,2;5,6-di-o-isopropylidene-d d-glucofuranose (5) could be an appropriate intermediate to synthesize d d-4-fluoro-3-thiophenol 12, which could be condensed with nucleobases to afford lamivudine analogues 1-4. diacetone glucose 5 was derived from d d-glucose by the known one-pot synthesis method reported by moravcova and his co-workers. 11 compound 5 was converted into 1,4-anhydro-4-thioarabitol 6 according to the known procedure developed by yoshimura and his co-workers over 10 steps. 12 surprisingly, benzoylation of compound 6 with benzoyl chloride, dmap, and pyridine at room temperature was very slowly converted to the corresponding benzoate 7. elevated temperature (88°c) completed the benzoylation reaction in 3 h in 94% yield. debenzylation of 7 with bcl 3 at à78°c gave a secondary alcohol 8 whose stereochemistry has the appropriate configuration to introduce a-fluoro substituent when treated with (diethylamino)sulfur trifluoride (dast). fluorination of 8 with dast gave the fluorinated compound 9 in 88% yield with the desired 'a' stereochemistry through double inversion mechanism by the nucleophilic participation of the ring sulfur atom. 13 removal of benzoyl group of 9 using cat. naome in meoh gave the corresponding alcohol 10 in nearly quantitative yield, which was used for coupling with benzoic acid under mitsunobu conditions. 14 during mitsunobu reaction the nucleophilic participation of sulfur atom occurred only to the extent of 3-5%. the major product 11 which underwent a single inversion was formed in 88% yield with its epimer (3-5%) at 3-position. the structures of major and minor product, 11 and its epimer, were confirmed by comparing 1 h nmr data of the corresponding alcohols generated from each epimer after debenzoylation with those of compound 10. debenzoylation of 11 afforded the glycosyl donor 12, which was ready for the condensation with nucleobases. synthesis of pyrimidine nucleoside analogues under mitsunobu conditions is illustrated in scheme 2. coupling of 12 with n 3 -benzoyluracil and n 3 -benzoylthymine, pph 3 , and dead gave the desired b-nucleosides 13 (62%) and 14 (66%), respectively. it is worthy to note that the participation of sulfur atom did not occur at all during coupling of glycosyl donor 12 with nucleobases. n-benzoyl group with 1 m naome produced tbdpsprotected compounds 15 and 16 in 82% and 94% yield, respectively. finally, treatment of 15 and 16 with tbaf gave the desired final compounds 1 and 2, respectively. confirmation of n-alkylated nucleosides 1 and 2 was determined by 13 c value of anomeric carbon of each nucleoside. 15 compound 1 was converted into lamivudine analogue, cytosine nucleoside 3, via acetylation of the hydroxyl group followed by successive three conventional steps (1,2,4-triazole, pocl 3 , pyridine; 1,4-dioxane, nh 4 oh; nh 3 , meoh) 16 in overall 54% yield. synthesis of adenine nucleoside analogue 4 is shown in scheme 3. whereas reaction of d d-4-fluoro-3-thiophenol 12 with n 6 -benzoyladenine at room temperature for 2 d under mitsunobu conditions (pph 3 and dead) did not give a clear spot expected as the desired product on tlc, reaction with 6-chloropurine as a nucleobase instead of n 6 -benzoyladenine afforded the protected 6chloropurine nucleoside 18 in 88% yield. participation degree of sulfur atom on the sugar ring seems like depending on electron density on the sugar ring, ring strain of the generated episulfonium cation, and properties of nucleophile and leaving group. mitsunobu reaction of fluorinated compounds (10 and 12) with benzoic acid or nucleobases might have difficulty in forming episulfonium cation because the sugar ring is already electron-poor by electron-withdrawing fluoro group. therefore, the major product was single inverted one (11, 13, 14, and 18 ). on the other hand, in case of dast reaction of compound 8 there is no strong electron-withdrawing group such as a fluorine atom on the sugar ring, implying that participation of the sulfur atom is expected and the major product is double inverted one (9). the remaining things are conversion of 6chloropurine into adenine base and desilylation of tbdps group in sequence. surprisingly, treatment of compound 18 with methanolic ammonia at 80°c overnight accomplished not only amination at 6-position of 6-chloropurine base but also desilylation of tbdps group, giving the final product 4 in 68% yield. antiviral activities of all iso-d d-2 0 ,3 0 -dideoxy-3 0 -fluorothianucleoside derivatives 1-4 synthesized as a bioisostere of lamivudine were measured against a variety of viruses such as hiv (human immunodeficiency virus) type 1 and 2, emcv (encephalomyocarditis virus), cox. b3 (coxsackie b virus type 3), vsv (vesicular stomatitis virus), and hsv (herpes simplex virus) type 1 and 2 (table 1 ). ec 50 of compounds 1, 2, and 4 exceeded 100 lg/ml and also did not show any cytotoxicity up to 100 lg/ml at all tested cells such as mt4, hela, and vero cells. on the other hand, cytosine nucleoside derivative 3 exhibited potent anti-vsv activity (ec 50 = 9 .43 lg/ml), whereas inactive (ec 50 > 100 lg/ ml) and non-cytotoxic (cs 50 and cc 50 > 100 lg/ml) against the other viruses. however, selectivity index (si) of 3 showed low value of 1.56 due to its high cytotoxicity (cc 50 = 14 .54 lg/ml) in hela cell. it is very interesting that cytosine nucleoside analogue 3 designed as a bioisostere of lamivudine showed anti-vsv activity instead of the expected anti-hiv activity. considering that vsv belongs to rna virus, it is worthy to note that 3 classified into ddns exhibited potent anti-vsv activity. antiviral evaluation of compounds 1-4 was also performed against flua (influenza virus type a) (h1n1) strain taiwan, flua (h3n2) strain johannesburg, fcv (feline coronavirus) strain wsu, and fip (feline infectious peritonitis virus) strain wsu, but all compounds did not show antiviral activity and cytotoxicity up to 100 lg/ml (not shown in table 1 ). we have designed and synthesized novel iso-d d-2 0 ,3 0 -dideoxythianucleoside derivatives 1-4 as a bioisostere of lamivudine to search for new anti-hiv agents. among compounds tested, only cytosine analogue 3 showed potent anti-vsv activity even if it pertains to ddns classification and vsv is rna virus. this observation implies that iso-d d-2 0 ,3 0 -dideoxy sugar templates might act as a sugar surrogate of nucleosides for the development of anti-rna virus agent. the information about participation of sulfur atom during fluorination reaction using dast and mitsunobu reaction will be of great help in synthesizing sulfur-containing compounds. melting points are uncorrected. ultraviolet (uv) spectra were recorded on a jasco v-530 uv/vis spectrophotometer. optical rotations were measured on a jasco dip-370 digital polarimeter. nmr data were recorded on a bruker ac200 and varian unity as 500 spectrometer, using cdcl 3 , or cd 3 od and chemical shifts were reported in parts per million (ppm) with reference to the respective residual solvent or deuteriated peaks (d h 3.30 and d c 49.0 for cd 3 od, d h 7.26 and d c 77.0 for cdcl 3 ). coupling constants are reported in hertz. the abbreviations used are as follows: s (singlet), d (doublet), q (quartet), m (multiplet), dd (doublet of doublets), br s (broad singlet), ddd (doublet of doublets of doublets), or td (triplet of doublets). fab mass spectra were recorded on jeol hx 110 spectrometer. elemental analyses were performed by the general instrument laboratory of ewha womans university, korea. all the reactions described below were performed under nitrogen or argon atmosphere and monitored by thin-layer chromatography (tlc). tlc was performed on merck precoated 60 f 254 plates. column chromatography was performed using silica gel 60 (230-400 mesh, merck). all anhydrous solvents were distilled over cah 2 or na/benzophenone prior to use. (+)-benzoic acid (3s,4s,5r)-4-benzyloxy-5-(tert-butyl-diphenyl-silanyloxymethyl)-tetrahydro-thiophen-3-yl ester (7) to a stirred solution of alcohol 6 (6.070 g, 12.68 mmol) and 4-dimethylaminopyridine (15 mg, 0.12 mmol) in anhydrous pyridine (30 ml) was dropwise added benzoyl chloride (1.62 ml, 13.96 mmol) at 0°c and the reaction mixture was heated to 88°c for 3 h. after the volatiles were removed under reduced pressure, the resulting residue was partitioned between ethyl acetate and 0.5 m hcl aqueous solution, and the organic layer was washed with saturated nahco 3 aqueous solution and brine successively, dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (15:1) as the eluent to give the corresponding benzoate 7 (6.927 g, 94%) as a colorless oil: ½a to a stirred solution of benzoate 7 (6.927 g, 11.89 mmol) in anhydrous ch 2 cl 2 (50 ml) was slowly added boron trichloride (28.5 ml, 28.50 mmol, 1 m solution in ch 2 cl 2 ) at à78°c and the reaction mixture was stirred at the same temperature for 40 min. pyridine (28.5 ml) and methanol (28.5 ml) were successively added and the reaction mixture was kept at room temperature for 1 h. the mixture was partitioned between methylene chloride and 0.5 m hcl aqueous solution, and the organic layer was washed with saturated nahco 3 aqueous solution and brine successively, dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (5:1) as the eluent to give the corresponding alcohol 8 (5.570 g, 95%) as a colorless oil: ½a to a stirred solution of alcohol 8 (3.050 g, 6.19 mmol) in anhydrous ch 2 cl 2 (20 ml) was dropwise added (dieth-ylamino)sulfur trifluoride (dast, 1.23 ml, 9.31 mmol) at à10°c and the reaction mixture was stirred at the same temperature for 30 min. after saturated nahco 3 aqueous solution was added, the reaction mixture was stirred for 5 min and partitioned between ch 2 cl 2 and water. the organic layer was dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (45:1) as the eluent to give the corresponding fluoro compound 9 (2.688 g, 88%) as a colorless oil: ½a to a stirred solution of fluoro compound 9 (2.655 g, 5.37 mmol) in methanol (7 ml) and ch 2 cl 2 (3 ml) was added 1 m naome (0.54 ml, 0.54 mmol) at 0°c and the reaction mixture was stirred for 4 h at room temperature. the mixture was partitioned between ch 2 cl 2 and water, dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (8:1) as the eluent to give the corresponding alcohol 8 (2.005 g, 96%) as a colorless oil: ½a to a stirred solution of alcohol 10 (1.360 g, 3.48 mmol), triphenyl phosphine (3.099 g, 11.82 mmol), and benzoic acid (1.440 g, 11.79 mmol) in anhydrous thf (20 ml) was dropwise added diethyl azodicarboxylate (1.9 ml, 12.07 mmol, dead) at 0°c over 5 min, and the reac-tion mixture was heated to 60°c for 5 h. after cooling, the reaction mixture was evaporated in vacuo. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (8:1 ! 6:1) as the eluent to give benzoate 11 (1.516 g, 88%) as a colorless oil: ½a 25 to a stirred solution of alcohol 12 (218 mg, 0.56 mmol), triphenyl phosphine (439 mg, 1.67 mmol), and n 3 -benzoyluracil (361 mg, 1.67 mmol) in anhydrous thf (6 ml) was dropwise added dead (0.26 ml, 1.65 mmol) at 0°c over 5 min, and the reaction mixture was stirred at room temperature overnight. after the volatiles were removed in vacuo, the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (3:1) as the eluent to give to a stirred solution of 13 (110 mg, 0.19 mmol) in methanol (4.5 ml) and ch 2 cl 2 (1.5 ml) was added 1 m naome (0.40 ml, 0.40 mmol, in meoh) at 0°c and the reaction mixture was stirred for 6 h at room temperature. the mixture was partitioned between etoac and water, dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was purified by silica gel column chromatography using hexane and ethyl acetate (1.5:1) as the eluent to give the uracil nucleoside 15 (74 mg, 82%) as a colorless sticky oil: ½a to a stirred solution of uracil nucleoside 15 (57 mg, 0.12 mmol) in thf (4 ml) was added 1 m tetrabutylammonium fluoride solution (0.34 ml, 0.34 mmol, tbaf, in thf) at 0°c, and the reaction mixture was stirred at room temperature for 2 h. after the reaction mixture was concentrated in vacuo, the resulting residue was purified by silica gel column chromatography using methylene chloride and methanol (15:1) as the eluent to give the final uracil nucleoside 1 (28 mg, 97%) as a colorless oil, which was crystallized in ether and a small amount of methanol to produce a white solid: mp 143.4-145.2°c; uv (meoh) k max 262 nm; ½a 13 to a stirred solution of uracil nucleoside 1 (18 mg, 0.07 mmol) in anhydrous pyridine (1.5 ml) was added acetic anhydride (0.02 ml, 0.21 mmol) at room temperature, and the reaction mixture was stirred at the same temperature overnight. the reaction mixture was evaporated in vacuo and partitioned between ethyl acetate and dilute hcl aqueous solution. the organic layer was washed with saturated nahco 3 aqueous solution, dried over mgso 4 , filtered, and evaporated under reduced pressure. the resulting residue was used to the next reaction without further purification. to a stirred suspension of the acetated residue 17 and 1,2,4-triazole (86 mg, 1.25 mmol) in anhydrous pyridine (2.5 ml) was added phosphorus oxychloride (0.08 ml, 0.83 mmol) at room temperature, and the reaction mixture was stirred at the same temperature for overnight. 1,4-dioxane (2 ml) and 28% ammonium hydroxide (2 ml) were added to the reaction mixture at 0°c and stirred at room temperature overnight. after the volatiles were evaporated under reduced pressure, methanolic ammonia (3 ml) was added to the resulting residue, and the reaction mixture was stirred at room temperature overnight. after the volatiles were removed in vacuo, the resulting residue was purified by silica gel column chromatography using methylene chloride and methanol (7:1) as the eluent to give the final cytosine nucleoside 3 (9 mg, 54%) as a light brownish solid, which was recrystallized in ether and a small amount of methanol: hygroscopic; uv (meoh) k max 271 nm; ½a cdcl 3 ) d 8.68 (br s, 1h, nh), 7.69-7.40 (m, 10h, 2· ar) 86 (s, 1h, chah-botbdps), 3.68-3.61 (m, 1h, 5-h), 3.15-3.10 (m, 2h, sch 2 ), 1.10 (s, 9h, t-bu); 13 c nmr (125 mhz (à)-1-[(3s,4s,5r)-5-(tert-butyl-diphenyl-silanyloxymethyl)-4-fluoro-tetrahydro-thiophen-3-yl]-5-methyl-1h-pyrimidine-2 14 mmol) was converted to 16 (65 mg, 94%) as a colorless sticky oil according to the similar procedure used in the preparation of compound 15: uv (chcl 3 ) k max 266 nm 05 (s, 1h, h-6) calcd for c 26 h 31 fn 2 o 3 ssi: c, 62.62 m, 1h, 3-h), 3.83 (dd, 1h, j = 6.5, 12.0 hz, ho-chahb), 3.71 (dd, 1h, j = 6.5, 11.5 hz, hochahb), 3.60-3.52 (m, 1h, 5-h), 3.30 (t, 1h, j = 11.0 hz, schahb), 3.04 (ddd, 1h, j = 1.0, 8.0, 11.0 hz, schahb); 13 c nmr (125 mhz, cd 3 od 17 mmol) was converted to 18 (78 mg, 88%) as a colorless sticky oil according to the similar procedure used in the preparation of compound 13: uv (chcl 3 ) k max 264 nm cdcl 3 ) d 8.75 (s, 1h, h-8) amino-purin-9-yl)-3-fluorotetrahydro-thiophen-2-yl]-cooling, the volatiles were removed in vacuo and the resulting residue was purified by silica gel column chromatography using methylene chloride and methanol (10:1) to give adenine nucleoside 4 (27 mg, 68%) as a colorless sticky oil, which was crystallized in ether and a small amount of methanol to afford a white solid: mp = 159 24 (s, 1h, h-2), 5.61 (ddd, 1h, j = 6.5, 8.5, 52.5 hz, 3-h), 5.39-5.31 (m, 1h, 4-h), 3.79 (dd, 1h, j = 6.0, 11.0 hz, hochahb), 3.72 (dd, 1 h, j = 6.0 11.0 hz, hochahb m+h) + ; anal. calcd for c 10 h 12 fn 5 os: c, 44 references and notes antimicrob. agents chemother key: cord-004733-i0a3igc7 authors: nagata, s.; okamoto, y.; inoue, t.; ueno, y.; kurata, t.; chiba, j. title: identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies date: 1992 journal: arch virol doi: 10.1007/bf01309581 sha: doc_id: 4733 cord_uid: i0a3igc7 thirteen monoclonal antibodies (mabs) to the glycoprotein (g) of vesicular stomatitis virus (vsv) serotype indiana were prepared and examined for their effects on various biological activities of vsv, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. competitive binding assays with these mabs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the g protein. in some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. the mabs to all the epitopes but one (epitopes 1–6) reacted with the denatured g protein in a western immunoblot analysis. four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. none of the mabs inhibited the adsorption of radiolabeled vsv to bhk-21 cells; the mabs to epitope 2 slightly enhanced the virus adsorption. all neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of vsv-mediated cell fusion. these results show a direct spatial relationship between the epitopes recognized by the mabs and functional sites on g protein and further insights into the structure and function of g protein. many enveloped viruses including vesicular stomatitis virus (vsv), family rhabdoviridae, genus vesiculovirus, transfer their nucleocapsids to the cytoplasm of host cells by the adsorption and receptor-mediated endocytosis, followed by fusion with the endosomal membrane [20, 21] . the glycoprotein (g) of vsv is the sole protein anchored in the viral envelope and plays a critical role in this early stage of virus infection. many biological properties of g protein are associated with the virus entry [37] , which include adsorption to host cells [3, 6] , hemagglutination (ha) [9, 23] , and mediation of in vitro cell-cell fusion [7, 38] . the cell-cell fusion occurs only at low ph, which mimics the acidic environment of the endosomal lumen [7, 38] . as expected from its central role in infection, the g protein also gives rise to and reacts with neutralizing antibodies [12] . in recent years, much effort has been made to reveal the structurefunction relationships of the g protein, especially regarding its role in fusion [2, 10, 27, 29, 39, 40] , but the underlying molecular mechanisms are still poorly understood. one approach to the structure-function relationship of surface glycoproteins of viruses is to analyze for the sites and effects of the monoclonal antibody (mab) binding [4, 13, 31, 33] . production of mabs against g proteins of two major serotypes of vsv (indiana and new jersey) has been reported by two research groups [5, 14, 15, 36] . these mabs have mainly been used to map neutralization and non-neutralization epitopes on g protein and to analyze the mutation leading to antigenic variations of g protein [8, 11, [16] [17] [18] 35] . the effects of the mabs specifically reacting with g protein on biological functions other than neutralization have not been reported. in the present study, we prepared thirteen mabs specific for seven distinct epitopes on g protein of vsv-indiana and examined for their effects on various biological activities of vsv including in vitro infection, ha, adsorption to the cells, and mediation of cell-cell fusion. our findings defined the spatial relationship between the epitopes recognized by the mabs and the functions of g protein. the san juan strain of vsv-indiana originally provided by dr. r. r. wagner, university of virginia, was obtained from dr. k. yamamoto, national institute of health, tokyo. the virus stock was prepared by infecting bhk-21 cells (japanese cancer research resources bank) at a multiplicity of 0.1 pfu/cell. virus harvested at 22 h postinfection was concentrated by ultrafiltration and ultracentrifugation [22, 25] , and purified by sucrose density gradient centrifugation [22] . this preparation containing 1.1 mg/ml of viral protein (2.3 x 1011pfu/ml) was stored at -80 °c. the protein content of the preparation was determined with bca protein assay reagent (pierce chemical co., rockford, il, u.s.a.) with bovine serum albumin (bsa) as a standard. virus infectivity was determined by plaquing on monolayer cultures of bhk-21 cells [41] . the g protein was extracted from the purified virus with 30 mm octyl-13-d-glucopyranoside (sigma chemical co., st. louis, mo, u.s.a.) as described by petri and wagner [30] . after removal of the nucleocapsids by ultracentrifugation at 150,000 x g, the supernatant containing g protein was dialyzed against 10 mm hepes (ph 7.4) containing 0.15 m naci. g protein thus obtained was free from any other virus protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) stained with coomassie brilliant blue. for immunization, female balb/c mice were subcutaneously injected twice each with 25 gg of the purified g protein emulsified in the same quantity of freund's complete and freund's incomplete adjuvants, respectively. then, additional two intraperitoneal injections with 40 gg of g protein were given. three days before fusion for hybridoma production, the final 40 gg of g protein was injected intravenously. the spleen cells of the immunized mice and sp 2/o-ag 14, balb/c mouse non-secretory plasmacytoma cells, were fused with polyethylene glycol according to oi and herzenberg [28] or by the novel vsv-mediated cell fusion method described previously [25, 26] . media preparation and hat selection of hybridomas were described previously [25] . in 2 weeks, the hybridomas were screened for production of anti-g protein antibody by enzyme-linked immunosorbent assay (elisa) with purified g protein as the antigen (see below). the positive cultures were cloned several times by the limiting dilution method. the isotypes of the specific antibodies were determined with a mouse monoclonal antibody isotyping kit (amersham international, buckinghamshire, england). thirteen hybridomas were established and each was over-grown in about 11 of a serumfree medium (iscove's modified dulbecco's medium, sigma) containing 1 mg/ml of bsa, 1 mm sodium pyruvate (gibco, grand island, ny, u.s.a.), 8 ~tg/ml of bovine insulin (sigma), 5 gg/ml of iron-saturated human transferrin (miles scientific, naperville, il, u.s.a.), 50 ~tm 2-mercaptoethanol, 20 gm ethanolamine, 2.5 ~tg/mt of linoleic acid, 2.5 ~tg/ ml of oleic acid, 2.5 gg/ml of palmitic acid, and 10 gg/ml of gentamicin. mab in the culture supernatant was precipitated with ammonium sulfate at 40% saturation and the precipitate was further purified by high performance liquid chromatography on hydroxyapatite beads [42] or by affinity chromatography on protein g-sepharose (pharmacia, uppsala, sweden). the eluate was concentrated to 4 ml by membrane ultrafiltration (30,000 tool. wt. cut-off centriprep; amicon, danvers, ma, u.s.a.), and dialyzed against phosphate-buffered saline (pbs). the antibody concentration was determined from absorbance at 280 nm with an extinction coefficient of 1.4 per mg of protein. for use as negative controls in various assays, two mabs prepared at national institute of health were purified by the same procedure. one of them was ig g1 specific for the core antigen of feline immunodeficiency virus (unpubl.) and the other was ig g2a specific for sheep red blood cells (not crossreactive with goose erythrocytes) [25] . production of anti-g protein antibody in hybridoma culture supernatants was examined by elisa. wells of microtiter plates (costar 3690; costar, cambridge, ma, u.s.a.) were coated with the purified g protein (1 gg/ml) in 50mm sodium carbonate buffer (ph9.6) for 2h at room temperature. the wells were washed with pbs containing 0.05% (v/v) tween 20 (pbs-tween) and blocked overnight at 4°c with 0.5% (w/v) gelatin in pbs. after washing, each culture supernatant was added to the wells and the plates were incubated for t h at room temperature. the antibody bound was detected by incubation for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse igg + m (tago 6553; tago inc., burlingame, ca, u.s.a.) diluted 5,000-fold in pbs-tween. the enzyme reaction was started by adding 1 mg/ml of p-nitrophenylphosphate (wako pure cemical ind., osaka, japan) in 1% (v/v) diethanolamine (ph 9.8) containing 0.5 mm mgc12. the 156 s. nagata et al. absorbance at 410 nm was measured with an eia autoreader (sanko junyaku co., tokyo, japan). as a positive control, a 2,000-fold dilution of mouse immune serum was used. this control usually showed an absorbance of approximately 1.0 after incubation for 20 rain at room temperature. the well with absorbance higher than 0.2 was regarded as positive. purified mabs were titrated by the same elisa to compare the relative reactivities, in which the wells of the plates were coated with vsv virions (2 gg/ml) instead of g protein. the relative reactivity was defined as the concentration of mab needed to attain 50% of the absorbance value of the positive control. purified vsv was separated by sds-page in 10% polyacrylamide gel under reduced conditions. the proteins separated were then blotted onto immobilone membrane (millipore corp., bedford, ma, u.s.a.) according to towbin et al. [34] . the blots were allowed to react with culture supernatants of established hybridomas, and specific bands were visualized with alkaline phosphatase-conjugated goat anti-mouse igg + m (tago 6553) and the bcip/ nbt phosphatase substrate system (kirkegaard & perry lab. inc., gaithersburg, md, u.s.a.). a 500-gg portion of each purified mab was mixed with 100gg of biotinyt n-hydroxysuccinimide ester (nhs-lc-biotin, pierce) in 0.5 ml of 0.1 m sodium bicarbonate (ph 8.4). after incubation for 4 h at room temperature, the mixtures were dialyzed extensively against pbs at 4 °c; bsa was then added to a final concentration of 5 mg/ml. for competitive binding assay, the wells of plates were coated with purified vsv (2 ~tg/ ml) and blocked with 5% (w/v) unfatted bovine milk in pbs. serial 10-fold dilutions of unlabeled competitor mab (2 x 10-1 to 2 x 10 -5 mg/ml) were added to the wells (40 ~tl/ well) and the plates were incubated for 3 h at room temperature. subsequently, 40 gl of biotinylated mab was mixed with competitor mab. the concentrations of biotinylated mabs were 1 gg/ml for 2b9 and 3b1, 0.5 gg/ml for v20 b12, and 0.25 gg/ml for the other mabs. these concentrations were about half-maximal in their titration curve and were within the range where the binding was linear. after incubation overnight at 4 °c, biotinylated mab bound was detected with a 5,000-fold dilution of alkaline phosphataseconjugated streptavidin (bethesda research lab., gaithersburg, md, u.s.a.). dilution was made in pbs-tween containing 5% (w/v) unfatted bovine milk. the enzyme reaction and absorbance determination were carried out as described above. all assays were performed in duplicate and the results were expressed as the percentage of binding calculated with the formula: average ofabsorbance in the presence of competitor binding(%) = x 100 average ofabsorbance in the absence of competitor for the neutralization assay, the stock of vsv was diluted to a final concentration of approximately 1,000 pfu/ml with bicarbonate-free eagle's minimum essential medium (mem, nissui pharmachemical co., tokyo, japan) containing 0.2 mg/ml of bsa and 20 mm hepes (ph 7.2). the virus was mixed with an equal volume of each of serial twofold dilutions of each purified mab (from 250 gg/ml) in the same medium. the mixtures were incubated for 1 h at 37 °c and then plated in duplicate on monolayers of bhk-21 cells in 6-well culture plates for plaque assay (100 gl/well). the neutralization antibody titer was defined as the reciprocal of the highest dilution reducing more than 50% of the plaques of the control without mab. hemagglutination inhibition (hi) was assayed with 4 or 8 hemagglutinating units of vsv in v-bottom microtiter plates as described by halonen et al. [9] , except that goose erythrocytes were used after the treatment with trypsin (sigma) at 100 l~g/ml for 30 rain at 37 °c. this pretreatment of the erythrocytes enhanced ha, thus increasing the sensitivity of hi [19; unpubt, data] . the reciprocal of the highest dilution of purified mab causing complete inhibition of hemagglutination was taken as the hi titer. for adsorption assays, 35s-labeled vsv was prepared by the addition of 20 ~ ci/ml of l-[35slmethionine (amersham) to the infection medium as described by bailey et al. [1] . the radiolabeled virions were concentrated and purified as described for the unlabeled virus. the final preparation was free of contaminating labeled materials as judged by sds-page and autoradiography. the final preparation contained 3.4 x 10 l° pfu/ml (1.8 mg/ml viral protein) with a specific activity of 1.5 x 104 cpm/~tg. the radiolabeled virus absorbed to cells was quantified essentially as described by matlin etal. [21] . bhk-21 cells grown to confluency in 12-well culture plates (about 10 6 cells/well) were washed twice with the binding medium, bicarbonate-free mem buffered with 20 mm hepes (ph 7.2) containing 2 mg/ml of bsa, and cooled for 5 rain on ice. the radiolabeled purified virus (33,500cpm) was mixed with each purified mab at various concentrations in the binding medium. the mixtures were incubated for 1 h at 37 °c, chilled, and then plated in duplicate on the bhk-21 cell (100 gl/well). after incubation for 1 h on ice, unbound virus was removed. the cells were washed four times with the binding medium. the cells bound with the virus were solubilized in 0.4 ml of solubable (nen research product, boston, ma, u.s.a.), and its radioactivity was measured with a liquid scintillation counter. the average radioactivities bound to the cells in the presence of mab were expressed as the percentage of the radioactivity bound in the absence of mab. nonspecific interaction of the virus with the ceils and the surface of the plates was minimized by adding bsa to the binding medium. the addition of bsa reduced the nonspecific binding of labeled vsv to the surface of the plates from 9.3% to less than 1.1% of the input radioactivity. the amount of virus used was within the range where the radioactivity bound to cells increased proportionally with the amount of the input virus. the effect of mabs on vsv-mediated cell fusion was assayed by inhibition of polykaryon formation of bhk-21 cells [24, 38] . the cells in 24-well culture plates (about 1.5 x 105 celts/well) for 18-24 h were washed twice with the ice-cold binding medium (bicarbonatefree mem buffered with 20 mm hepes, ph 7.2, containing 2 mg/ml of bsa). the purified virus (25 gg) in 200 gl of the cold binding medium was applied onto the cells. after incubation for 1 h on ice to allow viral adsorption, free virus was removed, and the cells were treated on ice for 45 min with 25 ~g/ml of each mab in 200 ~.1 of the binding medium. after removing the mab solutions, 0.5 ml of prewarmed (37 °c) acidic medium, bicarbonate-free mem buffered with 10 mm mes (ph 5.5), was added for triggering fusion and the plates were incubated for 2 min at 37 °c. the medium was replaced with 0.5 ml of the prewarmed binding medium and the cells were incubated for an additional hour at 37 °c. after fixation with 20% formalin in pbs and staining with hematoxylin, inhibition of polykaryon for-marion was examined under a phase-contrast microscopy. the amount of the virus used in this assay was enough to induce fusion in about 80% of the cells. two fusion experiments yielded 13 stable hybridomas secreting m a b s specifically reacting with g protein of vsv. their characteristics are listed in table 1 . their relative reactivities varied over a 40-fold range, but were still within a relatively high range c o m p a r e d with those of other m a b s to different antigens determined by us. all the m a b s except for p2f3 reacted with g protein in western blotting analysis. competitive binding e l i s a was carried out a m o n g these m a b s to classify the epitopes of g protein recognized by them. typical results o f the competitive binding assay are shown in fig. 1 , in which the binding o f biotinylated 1a7 m a b to g protein was challenged by several unlabeled mabs. we observed four types of competition. in addition to homologous m a b (1a7), p2f9 completely inhibited the binding. 5c6 partially inhibited the binding. 3f4, p2 e11 or p2f3 a all mabs had ~: light chain b relative reactivity was defined in elisa as the mab concentration needed to attain an absorbance of 0.6 when a positive control (immunized mouse serum diluted 2,000-fold) had an absorbance value of 1.2 c reactivity to g protein in western blotting analysis d epitopes (antigenic determinants) were identified by competitive binding assay among mabs as described in table 2 table 2 ). the 13 mabs were assigned to seven distinct epitopes on g protein based on the complete inhibition. when an unlabeled mab inhibited the binding of a biotinylated mab at 100gg/ml (at least 100-fold excess of biotinylated mabs) to less than 10% and when this inhibition was observed in pair-wise assays, both mabs were considered to share the same (or a closely adjacent) epitope. these seven epitopes were designated as ep 1 to ep 7. the mabs to the same epitope showed similar patterns of partial inhibition or enhancement of binding against mabs to different epitopes (table 2) . only mabs to ep 3 were subgrouped into two based on the pattern; mabs to ep 3a had no effect on the binding of mabs to ep 2, whereas mab to ep 3b enhanced the binding of mabs to ep 2. the 13 mabs were assayed for the neutralizing activity by the plaque reduction test ( table 3 ). the mabs assigned to four (ep 2, ep 4, ep 5 and ep 7) of the seven epitopes had neutralizing activities. the neutralization titers of the mabs to ep 5 and ep 7 were about 10-times higher than the others. inhibition of hemagglutination by the mabs was also examined ( table 3 ). the mabs assigned to two epitopes (ep 1 and ep 2) had higher hi activity than the others. goose erythrocytes used in this experiment were pretreated with trypsin to enhance the sensitivity of hi. untreated erythrocytes gave similar results, although higher mab concentrations were required (data not shown). titers for neutralization represent the reciprocal of the highest twofold dilution of purified mab (125 gg/ml initial) causing more than 50% reduction in the plaque number b titers for hi represent the reciprocal of the highest twofold dilution of purified mab (250 gg/ml initial) inhibiting ha caused by 4 or 8 hau of vsv. control ig g1 and ig g2a mabs and the serum-free medium for growing hybridomas showed no hi activity (< 1) we examined mabs for the influence on the adsorption of radiolabeled vsv to bhk-21 cells. mabs to ep 1 and ep 3 had very little, if any, effects on the virus adsorption (fig. 2 a and c) similar to the control mabs (fig. 2 h) . mabs to ep 4, ep 5, ep 6 and ep 7 slightly reduced the virus binding (fig. 2 d, e, f and g). even at the highest concentration (125 gg/ml), they exerted partial inhibition (65-75% of the binding of control). on the other hand, mabs to ep2 slightly enhanced the vsv adsorption only at certain concentrations (fig. 2 b) . in another experiment with a different preparation of radiolabeled vsv with a higher specific activity, similar results were obtained: no mab completely inhibited the virus binding and mabs to ep 2 enhanced the virus binding (data not shown). we examined mabs for the effects on vsv-mediated polykaryon formation of bhk-21 cells to test whether the mabs inhibit the fusion induced by vsv. extensive cell fusion was induced by acid treatment of the virus-bound cells (fig. 3 o) but not by the same treatment of unbound cells (fig. 3 p) . mabs reacting with ep 4 (fig. 3 g and h) , ep 5 ( fig. 3 i) , and ep 7 (fig. 31) completely inhibited polykaryocyte formation. the other mabs (fig. 3 a-f, j, and k) p2f3 + + --+ a + +, +, and -neutralization titers of 1> 1,000, >f 50 and > 1, respectively for 50% reduction of plaques, shown in the third column of table 3 b + and -hi titers against 4 h a u of >t 32 and ~< 16, respectively, given in the 4th column of table 3 c _ no inhibition or slight inhibition of adsorption (65-125%); e enhancement of adsorption (~> 135%) in the virus binding assay shown in fig. 2 d + inhibition of fusion; -no inhibition e not tested to the cell fusion activity. w e reported here for the first time identification of the g protein epitopes associated with h a and fusion activities. in the competitive binding assay, the m u t u a l complete competition o f paired m a b s revealed the presence of at least seven distinct epitopes on g protein of vsv-indiana. in addition, some topographical relationships a m o n g some o f these epitopes were suggested by partial inhibition or enhancement o f the binding o f m a b s ( table 2 ). in particular, association a m o n g ep 1, ep 2, and e p 3 w o u l d be quite possible since the m u t u a l binding enhancement o f the respective m a b s was observed. such enhancement is p r o b a b l y due to an a d v a n t a g e o u s allosteric alteration of g protein after binding with the first m a b , thereby resulting in increased binding o f the second m a b . similar competitive binding assays with anti-g protein m a b s were reported by volk etal. [36] and le-francois and lyles [14, 15] , w h o d e m o n s t r a t e d 11 and 10 epitopes on g protein of vsv-indiana, respectively. the enhancement of binding was f o u n d also by lefrancois and lyles [14, 15] . o f the seven epitopes identified on g protein, all b u t one (ep 1-6) reacted with respective m a b s even in western blot analysis. these are p r e s u m a b l y linear epitopes not dependent on the secondary structure. the m a b s assigned to four epitopes (ep 2, ep 4, ep 5, and ep 7) had vsv-neutralizing activity, although of varying efficiency. previous reports also demonstrated the same number of neutralizing epitopes on g protein of vsv-indiana [14, 36] . mabs to ep 1 and ep 2 showed hi activity. the proximity between these hi epitopes were suggested by the competitive binding assay and it is likely that these epitopes concurrently form the functional domain for the ha activity. these hi epitopes were not always neutralization epitopes and were different from the fusion-inhibition epitopes. this indicates that the sites involved in ha activity are different from those involved on the other functions. in general, viral ha is equivalent to the viral attachment to cells. however, the hi mabs did not inhibit the vsv binding to bhk-21 cells. the little correlation between these two activities is likely ascribed to the difference of the target cells and/or conditions in these assays. "l;he result of another experiment showed that the hi mabs markedly inhibit the binding of radiolabeled vsv to goose erythrocytes, in which the binding is measured under the same condition as the hi assay (data not shown). attempts to identify the cell-binding domain of g protein were unsuccessful in this study. in the binding assay, no mab completely inhibited the vsv adsorption (fig. 2) . mabs to ep 4, ep 5, ep 6, and ep 7 at high concentrations slightly inhibited vsv adsorption, but such low inhibitory effects were not related to the efficient neutralization or hi. the lack of the complete inhibition suggests that all neutralizing mabs prepared in this study block the virus infection at a step subsequent to adsorption. on the other hand, a certain concentration of mabs to ep2, one of the neutralization epitopes, rather enhanced vsv adsorption. although it is difficult to explain the biological significance of this enhancing effect, a similar enhancement of the vsv adsorption by immune serum was reported by schlegel and wade [32] . they suggested that the vsv-antibody complex binds to a different or an additional cell binding site, thus altering the adsorption efficiency. the same explanation may be given to the enhancing effect of mabs to ep 2. in the fusion inhibition assay, mabs reacting with three epitopes (ep 4, ep 5, and ep 7) inhibited the vsv-mediated cell fusion. these epitopes are probably located on or close to the fusogenic domain of g protein. although hydrophobic domains involved in fusion have been identified in several viral fusion proteins [10, 27] , such a domain has not been identified in g protein of vsv [27] . a most recent finding that introduction of a glycosylation site into residue 117 of g protein resulted in fusion-defective mutant suggests that the residues 118 to 136 are involved in the fusion activity [39] . some of our fusion-inhibiting mabs may recognize these residues and the location of the fusion-inhibition epitopes will be required. we found the presence of multiple epitopes related to the fusion activity. this suggests that these different regions of g protein contribute to the fusion activity in partnership and might explain the lack of highly hydrophobic fusion sequence in the g protein of vsv. all fusion-inhibiting mabs had the neutralizing activity. in the vsv infection process, after endocytosis of the b o u n d virion, the fusion of viral envelope with the endosomal m e m b r a n e is necessary for the entry of the nucleocapsids into the cytoplasm [20, 21] . fusion-inhibiting mabs neutralize vsv probably by blocking the fusion stage of the infection process. to analyze further the structure-function relationship of g protein, our studies are currently aimed at locating these epitopes on the g protein by testing the reactivity of the mabs to g protein fragments expressed in escherichia coli. effects of deae-dextran on infection and hemolysis by vsv. evidence that nonspecific electrostatic interactions mediate effective binding of vsv to cells glycosylation is not required for the fusion activity of the g protein of vesicular stomatitis virus in insect cells restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components identification of distinct antigenic determinants on semliki forest virus by using monoclonal antibodies with different antiviral activities monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (new jersey serotype): a method for preliminary mapping of epitopes surface structure of vesicular stomatitis virus a cell line expressing vesicular stomatitis virus glycoprotein fuses at low ph effect of glycosylation on the conformational epitopes on the glycoprotein of vesicular stomatitis virus (new jersey serotype) hemagglutinin of rabies and some other bullet-shaped viruses membrane fusion of envelope viruses: especially a matter ofproteins epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector the glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody biological activities of monoclonal antibodies reactive with antigenic sites mapped on the g1 glycoprotein of la crosse virus the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus i. analysis of neutralizing epitopes with monoclonal antibodies the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus ii. monoclonal antibodies to nonneutralizing and cross-reactive epitope of indiana and new jersey serotypes antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants point mutations in glycoprotein gene of vesicular stomatitis virus (new jersey serotype) selected by resistance to neutralization by epitope-specific monoclonal antibodies spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates ph-dependent hemolysis and cell fusion of rhabdoviruses virus entry into animal cells pathway of vesicular stomatitis virus entry leading to infection transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of madin-darby canine kidney cells. i. morphological evidence biological properties of vsv glycoprotein. ii. effects of the host cell and of the glycoprotein carbohydrate composition on hemagglutination vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different ph values production of monoclonal antibodies by the use of ph-dependent vesicular stomatitis virus-mediated cell fusion preferential generation ofmonoclonal igg-producing hybridomas by use of vesicular stomatitis virus-mediated cell fusion membrane fusion in fertilization, cellular transport, and viral infection immunoglobulin-producing hybrid cell lines vesicular stomatitis virus membrane proteins and their interactions with lipid bilayers functional epitopes on glycoprotein of vsv reconstitution into liposomes of the glycoprotein of vesicular stomatitis virus by detergent dialysis analysis ofmurine coronavirus surface glycoprotein functions by using monoclonal antibodies neutralized vesicular stomatitis virus binds to host cells by a different "receptor topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain jhm): correlation with biological activities electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications sequences of the major antibody binding epitopes of the indiana serotype of vesicular stomatitis virus monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity rhabdovirus biology and infection: an overview cell fusion by semliki forest, influenza, and vesicular stomatitis viruses a fusion-defective mutant of the vesicular stomatitis virus glycoprotein fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into vsv virions growth, purification and titration of rhabdoviruses. in: mahy bwj (ed) virology: a practical approach high performance liquid chromatography of mouse monoclonal antibodies on spherical hydroxyapatite beads we thank dr. kiichi yamamoto for the gift of vsv-indiana and drs. shudo yamazaki and robert r. wagner for their useful information on this strain. we thank also dr. yoshio yamakawa for his help with hplc and dr. akiko taniguchi for her assistance in some assays. several helpful discussions with dr. michiyuki matsuda are gratefully acknowledged. we thank also mr. john meissner and mr. russell nash for their critical reading of the manuscript. we thank also the members of department of pathology, national institute of health, for their encouragement. key: cord-296187-nnv2e7gr authors: mulgaonkar, nirmitee; wang, haoqi; mallawarachchi, samavath; fernando, sandun; martina, byron; ruzek, daniel title: bcr-abl tyrosine kinase inhibitor imatinib as a potential drug for covid-19 date: 2020-08-18 journal: biorxiv doi: 10.1101/2020.06.18.158196 sha: doc_id: 296187 cord_uid: nnv2e7gr the rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the infectious agent of coronavirus disease 2019 (covid-19) pandemic, poses an immediate need for potent drugs. enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. the sars-cov-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ace2) cell-surface receptor, is considered as a potential target for drug development. based on in silico screening followed by in vitro studies, here we report that the existing fda-approved bcr-abl tyrosine kinase inhibitor, imatinib, inhibits sars-cov-2 with an ic50 of 130 nm. we provide evidence that although imatinib binds to the receptor-binding domain (rbd) of sars-cov-2 spike protein with an affinity at micromolar, i.e., 2.32 ± 0.9 μm levels, imatinib does not directly inhibit the spike rbd:ace2 interaction – suggesting a bcr-abl kinase-mediated fusion inhibition mechanism is responsible for the inhibitory action. we also show that imatinib inhibits other coronaviruses, sars-cov, and mers-cov via fusion inhibition. based on promising in vitro results, we propose the abl tyrosine kinase inhibitor (atki), imatinib, to be a viable repurposable drug against covid-19. in early december 2019, the chinese health authorities reported several cases of pneumonia of unknown cause that had originated in wuhan, a city in the hubei province of china. the causative agent of this outbreak was identified to be a virus that belonged to the sarbecovirus subgenus, orthocoronavirinae subfamily which was previously referred to by its interim name 2019 novel coronavirus (2019-ncov) [1, 2] and was later named as sars-cov-2 [3] . due to the rapid spread of covid-19, the world health organization (who) declared it a global pandemic in march 2020 [4] . by mid-august 2020, over 21 million cases have been confirmed around the world, resulting in more than 768,000 deaths [5] . unfortunately, there is no approved antiviral treatment or preventive vaccine for coronaviruses in humans. since supportive care is the only recommended interim treatment, it is imperative to identify repurposable lead compounds to rapidly treat covid-19 patients until a sars-cov-2-specific drug and a vaccine is developed. although the coronavirus genome consists of numerous conserved druggable enzymes, including papain-like protease (plpro), 3c-like protease (3clpro), non-structural proteins rna-dependent rna polymerase (rdrp) and helicase, development of clinically approved antiviral therapies has proven to be a difficult task [6] . the surface structural spike glycoprotein (s), a key immunogenic cov antigen essential for virus and host cell-receptor interactions, is an important target for therapeutic development. the spike protein consists of an n-terminal s1 subunit (receptor binding) and a c-terminal s2 subunit (membrane fusion). the s1 subunit contains the receptor-binding domain (rbd) which attaches to the host membrane, thus playing an important role in viral entry. sars-cov-2 utilizes the ace2 receptor for entry and the transmembrane protease, serine 2 (tmprss2) for spike protein priming [7] . crystallographic studies have shown that sars-cov-2 binds to the ace2 receptor, with a binding mode nearly identical to that of sars-cov [8] [9] [10] [11] . the binding affinity of the ace2 receptor to the rbd of the sars-cov-2 spike protein is reported to be significantly higher as compared to sars-cov [10, 11] . based on the importance of virus membrane fusion events in the viral life cycle and its infectivity, the spike protein of sars-cov-2 was targeted for drug screening. this study utilizes in silico methodology followed by in vitro experimental validation to screen existing fda-approved small molecule drugs specific to the rbd of the spike protein of sars-cov-2 to identify repurposable drugs targeting further clinical validation. a model for sars-cov-2 spike protein was constructed using the crystal structure (6vsb_chain a) to correct missing residues. the amino acid sequence identity between the target sequence (genbank: qhd43416.1) and template (6vsb_chain a) was 99.58%. the sars-cov-2 model showed an rmsd of 0.4683 å relative to the crystal structure (6vsb_chain a). structure assessment of the predicted model using the ramachandran plot showed 90.04% residues in the most favored regions with 2.03% outliers. none of the outliers contained the residues present at the active site of the protein. the predicted model was further used for in-silico studies. in virtual screening, a library of approximately 5,800 compounds was docked against the sars-cov-2 spike rbd protein. the output was analyzed for common classes of drugs with highest (most negative) docking scores that resulted in seven compounds with three compounds, antiviral825, antiviral2038 and antiviral2981 with docking scores of -6.30 ± 0.00, -6.20 ± 0.20 and -6.00 ± 0.00 kcal/mol from the enamine antiviral library, and four compounds, ponatinib, imatinib, ergotamine, and glecaprevir with docking scores of -7.63 ± 0.06, -6.80 ± 0.17, -7.70 ± 0.00 and -7.20 ± 0.35 kcal/mol from the zinc15 fda library respectively. the above libraries were chosen to help identify a repurposable drug that can potentially inhibit the sars-cov-2. the screened compounds had the highest scores within their respective sets and had one or more binding conformations at the ace2 binding domain of the spike protein. the most common class of drugs was found to be abl tyrosine kinase inhibitors (atki), and hence two drugs (ponatinib and imatinib) with the highest scores were selected for in vitro testing. the binding scores for the seven screened compounds at the rbd are shown in fig. 1a and detailed description of the screened drugs is given in table s2 under supplementary data. the high affinity of the screened compounds is visible when compared with the negative control dimethyl sulfoxide (dmso), which is ineffective against coronaviruses [12] . based on promising in silico data, and initial viral plaque assay results (fig. 1a) , imatinib was chosen to be advanced for further experimental validation. (due to a supply-shortage ponatinib and ergotamine were unavailable for purchase and hence could not be included in the initial viral plaque assays). previous studies have shown imatinib to inhibit sars-cov and mers-cov by blocking endosomal fusion at the cell-culture level [6, [12] [13] [14] [15] . it has been suggested that tyrosinekinase inhibitors do not affect the cleavage of the spike protein but inhibit spike-mediated endosomal fusion [6, 12, 13] . the high affinity of tyrosine-kinase inhibitors towards the spike protein is deduced from the initial docking results, where both imatinib and ponatinib have shown highly negative binding free energies. first, we evaluated the toxicity of imatinib when incubating the compound on vero cells for one hour or eight hours. in the experiments where the compound remained on the cells for eight hours toxicity was measured at concentrations of 25 µm, 12.5 µm, 6.3 µm, and 3.2 µm. however, in the 1-hour design, no toxicity was observed. next, we evaluated the ability of imatinib to inhibit replication and entry. at concentrations as low as 0.2 µm the compound was effective in suppressing 50% of plaque formation in the 8-hr design, and the ic50 value determined using linear regression was 130 nm. consistent with the toxicity data, toxicity was observed between 25 and 3.2 µm. the compound also showed efficacy in the 1-hour design, with higher ic50 values. these data indicate that imatinib inhibits virus replication in vitro as shown in fig. 2a and 2b. to evaluate if imatinib inhibits viral entry, we performed two fusion assays: endosomal (vero) and plasma membrane (vero-tmprss2) as shown in fig. 2c and 2d, respectively. based on cytotoxicity, at concentrations below 15 nm, no toxicity was observed microscopically (red arrow in the graph). vsv-g control revealed 100% infectivity (cytopathic effect at every concentration below this, suggesting the inhibitor did not affect vsv-g entry. vsv-g particles cells do not carry spike proteins and thus, no significant entry inhibition occurred, suggesting that entry inhibition is likely mediated through the spike protein. however, the effect on vero-tmprss2 cells was less clear for any of the coronaviruses used when compared to the vsv-g control. a similar level of toxicity was observed in these cells. it is worth noting that toxicity is probably the result of incubating cells with imatinib for 16 hours in the assay. taken together, there is evidence that imatinib inhibits spike fusion and prevents viral entry, possibly by preventing endosomal entry. the binding kinetics of imatinib to the rbd of sars-cov-2 spike protein was evaluated using biolayer interferometry (bli), as shown in fig. 3a . the analysis showed that imatinib binds to the sars-cov-2 rbd protein with an on-rate (kon) as (3.22 ± 0.45) × 10 3 m -1 s -1 and dissociates with an off-rate (koff) as (7.07 ± 1.87) × 10 -3 s -1 . this resulted in an equilibrium affinity constant (kd) 2.32 ± 0.9 µm which is calculated as a ratio of the koff and kon rates. the affinity values indicate that 50% of the rbds on the surface spike glycoproteins will be occupied at micromolar concentrations of imatinib. however, this value is too close to the toxicity levels observed in the above assays and very high compared to the ic50 value, as well as the nanomolar affinity of ace2 on immobilized rbd (fig. s1 ) suggest that it is likely to inhibit spike fusion by the other previously suggested moa [12] . in vitro colorimetric assays were performed over a 160 pm to 500 nm range in imatinib concentration to assess the ability of imatinib to directly inhibit the rbd:ace2 interaction. the colorimetric signal of the positive control (no inhibitor) reaction was strong, and the blank wells exhibited an absorbance of ~ 0.05 at 450 nm as per the manufacturer's instructions. the test wells (with inhibitor) showed absorbance comparable to the positive control wells indicating that imatinib did not affect the sars-cov-2 rbd:ace2 interaction in the indirect competitive enzyme-linked immunosorbent assay (elisa), as shown in fig. 3b . a pharmacophore analysis was done to evaluate as to why imatinib showed promising in-silico results yet failed to directly inhibit the rbd:ace2 interaction. the primary binding site of the sars-cov-2 rbd was revealed via docking. pharmacophore analyses were done to further elucidate the interactions between the drug molecules and their receptors (fig. 4) . twenty-five pharmacophores were collected from the top five binding positions of imatinib at the primary active site of abl2 tyrosine kinase (native receptor), where each purple sphere represents a pharmacophore as depicted in fig 4a. similarly, an additional 25 pharmacophores were collected from the first five binding sites of imatinib at the primary active site of the sars-cov-2 rbd. the rbd pharmacophores were represented as yellow spheres in fig 4b. from the results of elixir-a alignment, it is evident that four pharmacophores between abl2 kinase and rbd overlapped (red spheres). this significant overlap reveals why the compounds that were originally screened using sars-cov-2 rbd also bound to abl2 kinase, ultimately ensuing in the inhibitory action. the above point is further explicated due to the 54.55% identity between the active sites of the abl2 (uniprotkb: p42684 aa 288-539) and sars-cov-2 spike rbd (uniprotkb: p0dtc2 aa 319-541) generated by a protein blast (blastp) [16, 17] search. there is an urgent need for finding a treatment against the current pandemic of the sars-cov-2. health experts across the globe are trying to use existing clinically approved drugs to treat patients until a specific drug is developed. the present study, using a combination of computational techniques followed by in vitro studies, identified imatinib, an fda approved anti-cancer drug as a potential treatment of sars-cov-2 infection. the data indicate inhibition of sars-cov-2 replication at ic50 of 130 nm. our results suggest that imatinib prevents viral replication by inhibiting the virus at the fusion stage, possibly by preventing endosomal entry. binding studies revealed that the affinity of imatinib for the sars-cov-2 spike rbd protein is still lower (higher kd value) than the previously published values of nanomolar range (ligand id: bdbm13530) [18] for imatinib on abl tyrosine kinase [19] and in range with the micromolar affinities of imatinib to the src-family kinases, frk and fyn [20] . although imatinib is not a promiscuous drug, it has been found to bind tightly to tyrosine kinases other than abl [20, 21] . pharmacophore mapping between abl2 and sars-cov-2 rbd and a 54.55% identity at the active site of the two proteins explains why imatinib binds to the sars-cov-2 rbd as well. however, imatinib failed to directly inhibit the sars-cov-2 spike rbd:ace2 interaction in the competitive elsa assays. therefore, it is likely that imatinib causes inhibition of virus fusion via cellular kinase pathway resulting in inhibition of virus replication, as previously described for other coronaviruses [12] . the results provide further evidence supporting the recent clinical trials (clinicaltrials.gov identifier: nct04346147, nct04357613, nct04356495, nct04394416, and nct04422678) for covid-19 patients with imatinib. a swiss-model server [22] was used to construct a homology model of the sars-cov-2 spike protein using the crystal structure of the sars-cov-2 spike protein (pdb:6vsb_chain a) as the template [10] . the genome sequence wuhan-hu-1 (genbank: mn908947.3) was used as a representative of the sars-cov-2. spike protein sequence (genbank: qhd43416.1) was used as the target sequence [23] . the swiss-model structure assessment tool was used to validate the quality of the predicted model. around 5,800 compounds, including 3,700 nucleoside-like compounds from the enamine targeted antiviral library (enamine.net) and 2,100 food and drug administration (fda)approved drugs from the zinc15 database [24] were used for molecular docking. all molecules were prepared with obabel [25] from .sdf or .mol2 format to .pdbqt format. the 3d compound structures from the enamine library were resolved by obabel --gen3d command. the docking file of the protein model was prepared with mgltools v1.5.4 [26] and the molecules were docked at the rbd of the spike protein via autodock vina 1.1.2 [27] . the grid box of 40×60×30 size with 1.0 å spacing was fixed around the rbd (thr323-val511) of the spike protein. each docking was done in three replicates, and the conformation with the highest binding score was recorded. the batch processing of docking and data collection was performed using an in-house python script which is deposited in github. data were analyzed statistically using r studio [28] and graphs were constructed with ggplot in r [29]. the ligand-receptor interactions were studied using schrödinger maestro [30] , and molecules with high docking scores were selected from each screening library for further studies. codon-optimized mers-cov (isolate emc, vg40069-g-n) and sars-cov (isolate cuhk-w1; vg40150-g-n) s expression plasmids (pcmv) were ordered from sino-biological and subcloned into pcaggs using the clai and kpni sites. the last 19 amino acids of the sars-cov spike protein were deleted to enhance pseudovirus production. codon-optimized cdna encoding sars-cov-2 s glycoprotein (isolate wuhan-hu-1) with a c-terminal 19 amino acid deletion was synthesized and cloned into pcagss in between the ecori and bglii sites. pvsv-egfp-dg (#31842), pmd2.g (#12259), pcag-vsv-p (#64088), pcag-vsv-l (#64085), pcag-vsv-n (#64087) and pcaggs-t7opt (#65974) were ordered from addgene. s expressing pcaggs vectors were used for the production of pseudoviruses, as described below. the cdna encoding human tmprss2 (nm_005656; ohu13675d) was obtained from genscript. the cdna fused to a c-terminal ha tag was subcloned into pqxcih (clontech) in between the noti and paci sites to obtain the pqxcih-tmprrs2-ha vector. vero-tmprss2 cells were produced by retroviral transduction. to produce the retrovirus, 10 μg pqxcih-tmprrs2-ha was co-transfected with polyethylenimine (pei) with 6.5 μg pbs-gagpol (addgene #35614) and 5 μg pmd2.g in a 10 cm dish of 70% confluent hek-293t cells in opti-mem i (1x) + glutamax. retroviral particles were harvested at 72 hours post-transfection, cleared by centrifugation at 2000 x g, filtered through a 0.45μm low protein-binding filter (millipore), and used to transduce vero cells. polybrene (sigma) was added at a concentration of 4 μg/ml to enhance transduction efficiency. transduced cells were selected with hygromycin b (invitrogen). hek-293t cells were maintained in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10% fetal bovine serum (fbs), 1x non-essential amino acids (lonza), 1mm sodium pyruvate (gibco), 2mm l-glutamine (lonza), 100 μg/ml streptomycin (lonza) and 100 u/ml penicillin. vero, vero-tmprss2, and veroe6 cells were maintained in dmem supplemented with 10% fbs, 1.5 mg/ml sodium bicarbonate (lonza), 10mm hepes (lonza), 2mm l-glutamine, 100 μg/ml streptomycin and 100 u/ml penicillin. all cell lines were maintained at 37°c in a 5% co2, humidified incubator. the protocol for vsv-g pseudovirus rescue was adapted from whelan and colleagues (1995). briefly, a 70% confluent 10 cm dish of hek-293t cells was transfected with 10µg pvsv-egfp-dg, 2µg pcag-vsv-n (nucleocapsid), 2µg pcag-vsv-l (polymerase), 2µg pmd2.g (glycoprotein, vsv-g), 2µg pcag-vsv-p (phosphoprotein) and 2µg pcaggs-t7opt (t7 rna polymerase) using pei at a ratio of 1:3 (dna:pei) in opti-mem i (1x) + glutamax. forty-eight hours post-transfection the supernatant was transferred onto new plates transfected 24 hours prior with vsv-g. after a further 48 hours, these plates were retransfected with vsv-g. after 24 hours the resulting pseudoviruses were collected, cleared by centrifugation at 2000 x g for 5 minutes, and stored at -80°c. subsequent vsv-g pseudovirus batches were produced by infecting vsv-g transfected hek-293t cells with vsv-g pseudovirus at a moi of 0.1. titres were determined by preparing 10-fold serial dilutions in opti-mem i (1x) + glutamax. aliquots of each dilution were added to monolayers of 2 × 10 4 vero cells in the same medium in a 96-well plate. three replicates were performed per pseudovirus stock. plates were incubated at 37°c overnight and then scanned using an amersham typhoon scanner (ge healthcare). individual infected cells were quantified using imagequant tl software (ge healthcare). all pseudovirus work was performed in a class ii biosafety cabinet under bsl-2 conditions at erasmus medical center. for the production of mers-cov, sars-cov, and sars-cov-2 s pseudovirus, hek-293t cells were transfected with 10 µg s expression plasmids. twenty-four hours post-transfection, the medium was replaced for in opti-mem i (1x) + glutamax, and cells were infected at a moi of 1 with vsv-g pseudotyped virus. two hours post-infection, cells were washed three times with optimem and replaced with medium containing anti-vsv-g neutralizing antibody (clone 8g5f11; absolute antibody) at a dilution of 1:50,000 to block remaining vsv-g pseudovirus. the supernatant was collected after 24 hours, cleared by centrifugation at 2000 x g for 5 minutes and stored at 4°c until use within 7 days. coronavirus s pseudovirus was titrated on veroe6 cells as described above. transduction experiments were carried out by incubating pseudovirus with imatinib at concentrations ranging from 0-125nm in opti-mem i (1x) + glutamax for 1 hour at 37°c. pseudovirus-imatinib mixes were added to monolayers of 2 × 10 4 vero or vero-tmprss2 cells in a 96-well plate. plates were incubated for 16 hours before quantifying gfp-positive cells using an amersham typhoon scanner and imagequant tl software. to determine the toxicity profile of imatinib, we performed the mtt assay using a 1-hr and an 8hr design. briefly, a serial dilution of imatinib was prepared and incubated on vero cells for 1 hr at 37 o c. subsequently, cells were washed, further cultured for eight hrs. in the 8-hr design, cells were incubated with a serial dilution of imatinib for eight hours without a washing step. we tested serial dilutions of imatinib for its ability to neutralize sars-cov-2 (german isolate; gisaid id epi_isl 406862; european virus archive global #026v-03883) using a plaque reduction neutralization test (prnt) as previously described [31] . fifty μl of the virus suspension (200 spot forming units) was added to each well and incubated at 37°c for either 1 hr. following incubation, the mixtures were added on vero cells and incubated at 37°c for either 1hr or 8 hrs. the cells incubated for 1 hr were then washed and further incubated in medium for 8 hrs. after the incubation, the cells were fixed and stained with a polyclonal rabbit anti-sars-cov antibody (sino biological; 1:500). staining was developed using a rabbit anti-sars-cov serum and a secondary alexa-fluor-labeled conjugate (dako). the number of infected cells per well were counted using the imagequant tl software. the binding kinetics of imatinib on sars-cov-2 rbd protein were studied using a blitz® system (fortébio). experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1x kinetics buffer (fortébio), 5% anhydrous dimethyl sulfoxide (dmso; sigma aldrich). recombinant his-tagged sars-cov-2 rbd protein (40592-v08h; sino biological) at a concentration of 10 µg/ml was loaded on anti-penta-his (his1k) biosensors (fortébio), followed by a washing step with assay buffer to block the unoccupied sensor surface. the association and dissociation profiles of imatinib (sigma aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µm to 0.78 µm). a reference biosensor loaded in the same manner with 0 µm imatinib was used for baseline correction in each assay. the final binding curves were analyzed with the blitz pro 1.3 software (fortébio) using the 1:1 global-fitting model. the assay was repeated twice to validate the binding constants. here, data is represented as mean ± sd. similarly, sars-cov-2 rbd was immobilized on his1k biosensors to study the binding kinetics of mfc-tagged hace2 (10108-h05h; sino biological) before being dipped into tubes containing the 1x kinetics buffer. various concentrations of ace2 (four-point serial dilutions from 800 to 100 nm) were used to measure the association and dissociation profiles. data were reference subtracted and fit to a 1:1 binding model using the blitz pro 1.3 software. the ability of imatinib to inhibit the interaction of spike rbd:ace2 proteins was evaluated by using the spike rbd (sars-cov-2): ace2 inhibitor screening colorimetric assay kit (bps bioscience). the avi-his-tagged spike s1 rbd (sars-cov-2) protein 50 ng/well in pbs was coated onto 96-well microplate by overnight incubation at 4°c. blocking buffer 2 was used to block the nonspecific binding sites by incubation for 1 hour. different concentrations of imatinib were added and incubated for 1 hour at room temperature with slow shaking. for the wells designated "blank" and "positive control", inhibitor buffer (pbs with 0.5% dmso) was added. the reaction was initiated by adding ace2 his-avi-tagged biotin-labeled hip tm protein (50 ng/well) in 1x immuno buffer 1 to the "positive control" and "test inhibitor" wells by incubation for 1 hour at room temperature with slow shaking. streptavidin-hrp (dilution 1:1,000 in blocking buffer 2) was added to each well and incubated at room temperature for 1 hour with slow shaking. washing procedure (3 × 100 µl 1x immuno buffer 1) was performed after each step. the chromogenic reaction was initiated by adding colorimetric hrp substrate to each well and incubated at room temperature until blue color was developed (approximately 2 minutes) in the "positive control" well. after the blue color was developed, the reaction was terminated by adding 1n hcl, and absorbance at 450 nm was measured using synergy h1 hybrid multi-mode microplate reader (biotek instruments). the pharmacophore model was generated using pharmit [32] , an online interactive platform to elucidate pharmacophores from the receptor and ligand complex. top five binding conformations of drug-protein complexes were produced by autodock vina. the pharmacophores of the ligands interacting with the receptor were considered active pharmacophores while the rest were defined as inactive pharmacophores. the pharmacophores from the native receptor of imatinib (abl2 tyrosine kinase; pdb:3gvu) and rbd of sars cov-2 were generated using imatinib as the ligand. using the enhanced ligand exploration and interaction recognition algorithm (elixir-a), the two sets of pharmacophores were merged and processed to identify any overlap in 3d space. a detailed description of elixir-a can be found in our previous work [33] and the algorithm has been deposited in github. the python script 'elixir-a-vina-batch-screening-module' used for running docking jobs in batch mode and elixir-a, the algorithm used for pharmacophore mapping have been deposited in github and will be made public upon publication of the manuscript. with md simulations. we are thankful to mart lammers for allowing us to use the fusion assays. funding: dr was supported by the ministry of health of the czech republic (project no. 20-05-00472). author contributions: sf, bm, and dr conceived and designed the study. first author nm designed the experiments, performed bli studies and immunoassays, reviewed literature, and compiled the manuscript and figures. co-first author hw conducted in silico experiments and compiled figures. sm did literature review on the resulting compounds and compiled the manuscript and figures. bm performed virology experiments. sf directed and verified studies and authored the manuscript. all authors reviewed and edited the paper. competing interests: all the authors declare that there are no conflicts of interest. percent inhibition compared to the amount of plaques on cells. here, 2-fold dilution of the compounds were done in duplo. then 300 tcid50 of sars-cov-2 was added to each well, and plates were incubated at 37c for 1 hour. then, the mixes were added onto vero cells and incubated for 8 hours at 37 c. subsequently, cells were fixed for 15 min with 2% pfa, followed by another 15 min fixation with 70% ethanol. fixed cells were stained with a monoclonal antibody, followed by alexafluor 488. here imatinib shows significant % inhibition as compared to other compounds tested. (1 µg/ml) were incubated for 1 hour at room temperature with slow shaking in the presence of various imatinib concentrations. streptavidin hrp (1:1,000) was added to the reaction mixture. colorimetric substrate was added to initiate the chromogenic reaction, and 2 minutes were allowed for color development. the reaction was terminated with the addition of 1n hcl and absorbance was measured at 450 nm. positive control (no inhibitor) was assumed to represent 0% inhibition. values obtained from test wells (with imatinib) compared to the positive control showed 0% inhibition of rbd:ace2 interaction, indicating that imatinib does not inhibit spike fusion by direct inhibition. pharmacophore distribution of five most stable conformations on tyrosine kinase abl2 (purple spheres); and b] pharmacophore distribution (yellow spheres) on sars-cov-2 spike protein rbd with pharmacophores common to both receptors depicted in red. a novel coronavirus from patients with pneumonia in china the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 who declares covid-19 a pandemic an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases coronaviruses-drug discovery and therapeutic options sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor functional assessment of cell entry and receptor usage for lineage b β-coronaviruses discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. biorxiv cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion corona virus drugs -a brief overview of past, present and future repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection gapped blast and psi-blast: a new generation of protein database search programs protein database searches using compositionally adjusted substitution matrices. the febs journal bindingdb: a web-accessible database of experimentally determined protein-ligand binding affinities. nucleic acids research a quantitative analysis of kinase inhibitor selectivity a small molecule-kinase interaction map for clinical kinase inhibitors molecular therapeutics: is one promiscuous drug against multiple targets better than combinations of molecule-specific drugs? swiss-model: homology modelling of protein structures and complexes complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in wuhan docking.org: over 1.5 billion compounds you can search and buy; 550 million leadlike you can dock. abstracts of papers of the open babel: an open chemical toolbox autodock4 and autodocktools4: automated docking with selective receptor flexibility software news and update autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading gplots: various r programming tools for plotting data. 2015. 30. release, s., 1: maestro. schrödinger, llc severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease pharmit: interactive exploration of chemical space a non-beta-lactam antibiotic inhibitor for enterohemorrhagic escherichia coli o104: h4 pharmacophore distribution on sars-cov-2 spike protein rbd with imatinib we gratefully acknowledge the support from texas a&m high performance research computing (hprc) and tamu laboratory for molecular simulation (lms). we would like to thank dr. lisa perez (associate director for advanced computing enablement, hprc tamu) for guidance key: cord-001765-7wv4cb37 authors: matassov, demetrius; marzi, andrea; latham, terri; xu, rong; ota-setlik, ayuko; feldmann, friederike; geisbert, joan b.; mire, chad e.; hamm, stefan; nowak, becky; egan, michael a.; geisbert, thomas w.; eldridge, john h.; feldmann, heinz; clarke, david k. title: vaccination with a highly attenuated recombinant vesicular stomatitis virus vector protects against challenge with a lethal dose of ebola virus date: 2015-06-24 journal: journal of infectious diseases doi: 10.1093/infdis/jiv316 sha: doc_id: 1765 cord_uid: 7wv4cb37 previously, recombinant vesicular stomatitis virus (rvsv) pseudotypes expressing ebolavirus glycoproteins (gps) in place of the vsv g protein demonstrated protection of nonhuman primates from lethal homologous ebolavirus challenge. those pseudotype vectors contained no additional attenuating mutations in the rvsv genome. here we describe rvsv vectors containing a full complement of vsv genes and expressing the ebola virus (ebov) gp from an additional transcription unit. these rvsv vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rvsv/human immunodeficiency virus type 1 vaccine. one of these rvsv vectors (n4ct1-ebovgp1), which expresses membrane-anchored ebov gp from the first position in the genome (gp1), elicited a balanced cellular and humoral gp-specific immune response in mice. guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal ebov challenge, while control animals died in 7–9 days. subsequently, n4ct1-ebovgp1 demonstrated complete, single-dose protection of 2 macaques following lethal ebov challenge. a single sham-vaccinated macaque died from disease due to ebov infection. these results demonstrate that highly attenuated rvsv vectors expressing ebov gp may provide safer alternatives to current ebov vaccines. previously, recombinant vesicular stomatitis virus (rvsv) pseudotypes expressing ebolavirus glycoproteins (gps) in place of the vsv g protein demonstrated protection of nonhuman primates from lethal homologous ebolavirus challenge. those pseudotype vectors contained no additional attenuating mutations in the rvsv genome. here we describe rvsv vectors containing a full complement of vsv genes and expressing the ebola virus (ebov) gp from an additional transcription unit. these rvsv vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rvsv/human immunodeficiency virus type 1 vaccine. one of these rvsv vectors (n4ct1-ebovgp1), which expresses membrane-anchored ebov gp from the first position in the genome (gp1), elicited a balanced cellular and humoral gp-specific immune response in mice. guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal ebov challenge, while control animals died in 7-9 days. subsequently, n4ct1-ebovgp1 demonstrated complete, single-dose protection of 2 macaques following lethal ebov challenge. a single sham-vaccinated macaque died from disease due to ebov infection. these results demonstrate that highly attenuated rvsv vectors expressing ebov gp may provide safer alternatives to current ebov vaccines. keywords. attenuation; rvsv vector; ebola vaccine; glycoprotein; challenge; nonhuman primates. the genus ebolavirus is classified within the filoviridae family and comprises 5 closely related but antigenically distinct virus species: bundibugyo ebolavirus, reston ebolavirus, sudan ebolavirus, tai forest ebolavirus, and zaire ebolavirus [1, 2] . in nature, all but reston virus have been isolated only in sub-saharan africa, where the likely virus reservoir is thought to be in bats [3, 4] . all but the tai forest and reston viruses have caused sporadic, deadly outbreaks of disease in humans since the discovery of ebolavirus 38 years ago, with case-fatality rates of 30%-90% [5, 6] . the ongoing 2014 ebola virus (ebov) outbreak in west africa is the largest recorded, with case-fatality rates of around 50%. hospitalization of patients seemingly improves clinical outcome and public health initiatives may slow the spread of disease, but mass vaccination programs in ebov-endemic regions will likely be required to extinguish the current outbreak and prevent such occurrences in the future. the ebov genome is composed of a single strand of negative-sense rna, approximately 19 kb in length, encoding 7 major viral polypeptides. one of these proteins is the ebov glycoprotein (ebovgp), which mediates virion attachment and fusion to susceptible cells [7] . the ebovgp also contains the major virus neutralization epitopes [8, 9] ; accordingly, the ebovgp has been the target for a variety of ebov vaccine designs. for a thorough review of vaccine approaches see falzarano et al [10] . the vector in one such vaccine is recombinant vesicular stomatitis virus (rvsv), in which the gene encoding vsv g has been replaced with the gene encoding ebovgp [11, 12] . this rvsv pseudotype replicates efficiently in cell culture and also propagates in vivo. vsv is a member of the rhabdoviridae family and, similar to ebov, has a single-strand, negative-sense rna genome, approximately 11 kb long, encoding 5 major viral proteins. in nature, vsv cycles between biting insects, which are the likely reservoir, and livestock [13] [14] [15] . the virus replicates to high titer in virus-induced vesicles at insect bite sites on the nose, lips, teats, and coronary bands of hooves. disease in livestock is self-limiting and typically resolves in 10-11 days without significant consequences. vsv can also infect humans who have close contact with infected animals or purified virus [16, 17] . infection results in mild influenza-like symptoms, which typically resolve in 3-5 days without complications [16, 17] . the very low seroprevalence of vsv in humans favored development of vsv as a vaccine vector and became feasible when rose et al developed a system for recovery of rvsv from genomic complementary dna (cdna) [18] . since then, rvsv vectors expressing antigens from human pathogens have demonstrated high levels of efficacy in the respective animal disease models [19] [20] [21] [22] . vsv exhibits a pronounced 3′ to 5′ gradient of gene expression due to discontinuous messenger rna (mrna) transcription across intergenic regions [23, 24] , allowing modulation of virus protein or foreign antigen expression as a function of gene distance from the 3′ transcription promoter [25, 26] . a highly attenuated rvsv vector expressing human immunodeficiency virus type 1 (hiv) gag from the first position in the genome was developed and has been demonstrated to be safe in stringent mouse and nonhuman primate (nhp) neurovirulence and biodistribution studies [27] [28] [29] . this rvsv vector was attenuated by combining n gene translocation from position 1-4 (n4) in the genome and truncation of the g protein cytoplasmic tail (ct) from 29 amino acids (aa) in native form to 1 aa (ct1) [28, 30] . this n4ct1-hivgag1 vector was subsequently shown to be safe and immunogenic in phase 1 clinical trials (hvtn 090 and hvtn 087; available at: http://clinicaltrials. gov/), and after vaccination viremia was undetectable by an vsv infectivity assay or a vsv-specific polymerase chain reaction assay in blood, urine, or saliva specimens obtained from trial participants (unpublished data). here we describe the generation of attenuated rvsv vectors expressing ebovgp and their immunogenicity in mice. the vector that elicited the most balanced ebovgp-specific humoral and cellular immune response in mice was then evaluated for protective efficacy in guinea pig and nhp challenge studies. for murine studies, 8-10-week-old female balb/c mice were used. mice were maintained according to the guide for the care and use of laboratory animals [31] . in addition, procedures for the use and care of the mice were approved by profectus biosciences and new york medical college institutional animal care and use committees (iacucs). the guinea pig and macaque studies were carried out in strict accordance with the recommendations described in the guide for the care and use of laboratory animals of the national institutes of health, the office of animal welfare, and the us department of agriculture. all animal work was approved by the national institute of allergy and infectious diseases division of intramural research iacuc at the rocky mountain laboratories (rml). the facility is accredited by the american association for accreditation of laboratory animal care. all procedures were performed on animals after they were anesthetized by trained personnel under the supervision of veterinary staff. all efforts were made to minimize the pain and suffering of animals, with early end point criteria specified by the rml iacuc-approved score parameters used to determine when animals should be humanely euthanized. as described previously [28, 30] , the n4ct1-hivgag1 vector was used as template for generating all rvsv/ebov vectors expressing ebovgp. the n4ct1-hivgag1 genomic cdna was modified by exchanging the gag gene located in position 1 of the genome with the ebovgp gene (1976, mayinga isolate), generating n4ct1-ebovgp1 cdna (gp1; figure 1a) . a vector expressing a third-position ebovgp (gp3; figure 1a ) was generated by inserting an expression cassette between the m and n genes. to insert the ebovgp gene in genome position 6 (gp6; figure 1a ), the n and g genes were swapped and an expression cassette was positioned between vsv l and the trailer sequence. for adventitious agent testing of clinical trial material, large quantities of ebovgp-specific neutralizing antibody are required to neutralize vaccine virus infectivity. to avoid this requirement, a gene expressing secreted ebovgp was generated by deleting the sequence encoding the transmembrane region and cytoplasmic tail (aa 651-676). this modified ebovgp gene was inserted into position 1 of n4ct1 (gp1δtm; figure 1a ). the mucin-like domain of ebovgp spans aa 305-485, and part of this domain (aa 374-465) was deleted (gp1δmuc; figure 1a ). this modified ebovgp gene was generated following observations that part of the mucin region was deleted upon multiple serial passage of n4ct1-ebovgp1 on vero cell monolayers and was included here to assess the effect of partial removal of the glycan "coat" on ebovgp immunogenicity. ebovgp expression was analyzed by western blotting for all vectors ( figure 1b ). vectors were rescued from genomic cdna as previously described [32] . rescued virus was plaque purified and amplified on vero cell monolayers (atcc ccl-81). for animal studies, vectors were purified from infected cell supernatants by centrifugation through a 10% sucrose cushion. virus pellets were resuspended in phosphate-buffered saline (pbs), ph 7.0, mixed with stabilizer (7 mm k 2 hpo 4 , 4 mm kh 2 po 4 , and 218 mm sucrose), snap frozen, and stored at −80°c. for the mouse immunogenicity study, rvsv/ebov vectors were administered at 1 × 10 7 plaque-forming units (pfu) by intramuscular injection of the tibialis anterior muscle (total injection volume, 0.05 ml). serum was collected at weeks 0, 3, and 5. ten days after final the inoculation, mice were euthanized by co 2 inhalation, and serum and spleen cells were harvested. for the mouse neurovirulence study, groups of ten 5-weekold female swiss webster mice were anesthetized and inoculated intracranially with 0.02 ml of serial 10-fold dilutions of each vector as previously described [30] . after inoculation, mice were weighed daily and assessed for signs of disease over 3 weeks. neurovirulence was assessed by measuring morbidity and mortality as an end point. mice that were showing severe signs of disease or were moribund were promptly euthanized. cumulative deaths across all doses tested allowed the 50% lethal dose to be calculated for each vector [33] . for guinea pig immunization, 2 × 10 5 pfu of each vector was administered intraperitoneally. on day 21 after vaccination guinea pigs were challenged with 10 focus-forming units (ffu) of guinea pig-adapted ebov (gpa-ebov) [34] by intraperitoneal injection. all animals were weighed daily and monitored for signs of illness. for macaque challenge, 3 healthy, ebov-naive, adult (age, 5-10 years; weight, 3-12 kg), male and female rhesus macaques (macaca mulatta) were randomly assigned to a vaccination group (n = 2) and a nonvaccination control group (n = 1). at study day −21, macaques were anesthetized with ketamine and inoculated in the caudal thigh muscle with 1 × 10 7 pfu of rvsv/ebov vector (total volume, 1 ml). at study day 0, macaques were anesthetized with ketamine and challenged with 1000 ffu of ebov [11] by intramuscular injection of the caudal thigh muscle (total volume, 1 ml). macaques were monitored for signs of illness (ie, abnormal temperature, weight loss, clinical examination findings, hematology findings, and blood chemistry findings) following vaccination and the ebov challenge portions of the study. for murine studies, vaccine-elicited ifn-γ elispot responses were determined using a mouse ifn-γ elispot kit (bd biosciences, san diego, california) as previously described [35] . splenocytes were incubated with 2 µg/ml con-a (sigma), peptide pools (15-mers overlapping by 11 aa; final peptide concentration, 1 µm [each]) spanning ebovgp, or medium alone. a murine enzyme-linked immunosorbent assay (elisa) previously described [35] was modified by using elisa plates coated with 100 ng/well of purified recombinant ebovgp (ibt bioservices) to determine ebovgp-specific serum immunoglobulin g (igg) titers. murine serum samples were added to elisa plates at a starting dilution of 1:100 and were further diluted 3-fold across the plates. antigen-specific antibody titers were defined as the reciprocal of the last serum dilution giving an od 450 of >0.1. antigens for the macaque elisa were produced as previously described [36] . macaque sera were inactivated by γ-irradiation (5 mrad) according to a standard operating protocol approved by the institutional biosafety committee at rml. elisa with ebovgpδtm was performed as described previously [36] . to identify the most-effective rvsv/ebov vector design for induction of ebovgp-specific immune responses, the vectors outlined in figure 1a were compared for their ability to elicit ebovgp-specific cell-mediated immune and binding antibody responses in mice (figure 2 ). groups of balb/c mice (n = 10) were immunized intramuscularly at study week 0 ( figure 2a ). ten days after primary immunizations, splenocytes were collected from 5 mice/group and tested for ebovgp-specific ifn-γ secretion by elispot assay. the remaining 5 mice/ group were boosted intramuscularly at study week 3 with 10 7 pfu of each rvsv/ebov vector. ten days after boosting, splenocytes were collected and tested as described above. after 1 immunization, the highest mean ebovgp-specific ifn-γ elispot responses (±standard error of the mean [sem]) were detected in mice immunized with n4ct1-ebovgp1δtm (215 ± 25 spot-forming cells [sfcs]/10 6 splenocytes) and n4ct1-ebovgp1 (133 ± 17 sfc/10 6 splenocytes). interestingly, n4ct1-ebovgp1δmuc, elicited a significantly reduced response (mean ± sem, 43 ± 19 sfcs/10 6 splenocytes) relative to n4ct1-ebovgp1 and n4ct1-ebovgp1. notably, after 1 immunization, gp responses were undetectable in mice immunized with vectors expressing ebovgp from genome positions 3 or 6. after boosting, the highest mean ebovgp-specific ifn-γ elispot responses (±sem) were again detected in mice immunized with n4ct1-ebovgp1δtm (211 ± 18 sfcs/10 6 splenocytes) and n4ct1-ebovgp1 (mean ± sem, 300 ± 57 sfcs/10 6 splenocytes). mice immunized with n4ct1-ebovgp1δmuc showed an improved response (mean ± sem, 172 ± 41 sfcs/ 10 6 splenocytes), and mice boosted with n4ct1-ebovgp3 or n4ct1-ebovgp6 still failed to demonstrate a measurable ebovgp-specific ifn-γ elispot response. immunized mice were also monitored for induction of serum antibody responses by elisa ( figure 2b ). ten days after boosting, mean serum ebovgp-specific igg titers (±sem) were highest in mice immunized with n4ct1-ebovgp1δmuc vector (5.35 ± 0.08), n4ct1-ebovgp1 (5.19 ± 0.07), and n4ct1-ebovgp3 (5.14 ± 0.07). interestingly, a statistically significant, approximately 10-fold lower ebovgp-specific igg titer was detected in mice immunized with n4ct1-ebovgp1δtm (mean ± sem, 4.05 ± 0.25) relative to mice immunized with n4ct1-ebovgp1. a 10-fold lower titer was also detected in mice immunized with n4ct1-ebovgp6 (4.21 ± 0.22) relative to mice immunized with vectors expressing ebovgp from genome positions 1 or 3. on the basis of the data presented in figure 2 , the n4ct1-ebovgp1 vector expressing an anchored full-length ebovgp in position 1 of the rvsv genome appeared to be the most immunogenic (with regard to cell-mediated and humoral immunity) in mice, so n4ct1-ebovgp1 was selected for guinea pig and nhp challenge studies. we next tested whether n4ct1-ebovgp1 could protect guinea pigs from a lethal challenge (figure 3 ). in this experiment, guinea pigs were immunized intraperitoneally with 2 × 10 5 pfu of n4ct1-ebovgp1. for controls, additional groups of guinea pigs were immunized with 2 × 10 5 pfu of wtvsv or medium alone. on day 21 after immunization, all guinea pigs were challenged intraperitoneally with 10 ffu of gpa-ebov. after challenge, guinea pigs were monitored for changes in body weight ( figure 3a) , signs of disease, and survival ( figure 3b ). guinea pigs immunized with medium alone or wtvsv began to lose weight on day 4 after challenge and died from ebov infection during study days 7 and 9 as observed previously [37] . in contrast, n4ct1-ebovgp1 immunized guinea pigs gained weight and were completely protected from disease during the course of the study. encouraged by findings from the guinea pig study, we determined whether n4ct1-ebovgp1 could protect macaques from lethal ebov challenge (table 1) . three rhesus macaques were immunized intramuscularly in the caudal thigh muscle as follows: 2 were immunized with 1 × 10 7 pfu n4ct1-ebovgp1 in a total volume of 1 ml (ebov1 and ebov2), and 1 macaque (ctrl) was mock immunized. at day 14 after immunization, both n4ct1-ebovgp1-immunized macaques demonstrated measurable ebovgp-specific serum igg responses (titer, 1:100), and these responses increased (titer, 1:400) by study day 0. in contrast, the sham-immunized macaque remained seronegative for ebovgp. at study day 0, all 3 macaques were challenged with 1000 ffu ebov mayinga by intramuscular injection in the caudal thigh muscle. as shown in table 1 , the single control animal failed to mount an ebovgp-specific response on day 6 after challenge and quickly died from ebov infection. importantly, a rapid increase in the ebovgp-specific serum igg titer was observed on days 6, 10, and 14 after challenge in both vaccinated macaques, and they survived without showing signs of disease. mice are exquisitely sensitive to intracranial instillation of wtvsv [28, 30] , leading to lethal viral encephalitis. previously, it was shown that intracranial inoculation of 5-week-old swiss webster mice with 10 8 pfu of the highly attenuated n4ct1-hivgag1 vector caused no significant illness, so a 50% lethal dose could not be achieved for this vector [28] . to test the neurovirulence potential of n4ct1-ebovgp1, groups of outbred swiss webster mice were inoculated intracranially with n4ct1-ebovgp1 in a dose-ranging study (10 4 -10 7 pfu; figure 4 ). two control groups were inoculated with 10 2 pfu of a less attenuated rvsv (rvsv-hivgag5) or with pbs alone. following inoculation, mice were assessed for morbidity and mortality over 2 weeks. consistent with previously published reports [28, 30] , mice inoculated intracranially with pbs survived, and approximately half of the mice inoculated with rvsv-hivgag5 died 3-5 days after inoculation. importantly, all mice inoculated with n4ct1-ebovgp1 survived and were disease free. previous work demonstrated that vaccination with a single dose of replication-competent rvsv pseudotyped with ebovgps protected mice, hamsters, guinea pigs, and nhps from lethal ebov challenge [12, 38, 39] . the rvsv vector used in those studies retained the natural vsv genome organization, except the vsv g gene was replaced with the ebovgp gene [39] . here we demonstrate protection of guinea pigs and nhps with a highly attenuated, replication competent form of rvsv that retains the vsv g gene [27, 28, 30] and expresses the ebovgp from an additional transcription unit inserted at position 1 in the rvsv genome. the mouse immunogenicity study demonstrated that ebovgpspecific cell-mediated immune responses were more robust when the ebovgp gene was in the first position in the genome, compared with the third and sixth positions, consistent with greater relative abundance of ebovgp in infected cells due to the steep 3′ to 5′ transcription gradient present during vsv replication [23, 24, 26] . the cell-mediated immune response was similar for secreted and anchored forms of ebovgp, indicating that both forms of ebovgp underwent a similar degree of processing and presentation of t-cell epitopes in antigen-presenting cells. interestingly, there was a significant reduction in the cell-mediated immune response to ebovgp when part of the mucin-rich region was deleted, possibly as a result of altered proteosomal processing of ebovgp or direct deletion of t-cell epitopes. after decay of the primary cell-mediated immune response, a second vaccine dose boosted elispot responses to levels seen after primary vaccination; however, there was still no detectable cell-mediated immune response elicited by vectors expressing ebovgp from position 3 and 6, indicating the requirement for higher ebovgp levels in infected cells to generate detectable ebovgp-specific cell-mediated immune responses. as expected, the ebovgp-specific igg response in mice was greatest for n4ct1-ebovgp1 and n4ct1-ebovgp1δmuc vectors expressing the highest levels of ebovgp from genome position 1. interestingly, n4ct1-ebovgp1δtm elicited much lower levels of ebovgp-specific igg than n4ct1-ebovgp1. we speculate this may reflect differences in the tertiary and quaternary structure of secreted ebovgp, compared with the trimeric conformation achieved by anchored ebovgp. notably, the secreted ebovgp described here is different from the naturally occurring secreted form of ebovgp that is translated from unedited ebovgp mrna [40] . the corollary is that anchored trimeric ebovgp spikes on the surface of infected cells and virus particles present a dense array of structural epitopes that may interact more efficiently with b cells than soluble secreted ebovgp, as is the case for other particulate antigen compositions [41, 42] . ebovgp-specific igg responses were highest for n4ct1-ebovgp1δmuc [43] , possibly reflecting better exposure of ebovgp epitopes by removal of part of the carbohydrate cloak [44] . the n4ct1-ebovgp3 vector also elicited a robust ebovgp-specific igg response, which contrasts with the undetectable ebovgp-specific cell-mediated immune response induced by this vector. the explanation for this anomaly is unclear but could be due to relative differences in ebovgp-specific cellular and humoral antigenicity. we believe the approximate 30% attenuation of expression across each gene junction could drop the ebovgp level below a threshold required to elicit cell-mediated immune responses (n4ct1-ebovgp3), while the ebovgp level is still adequate to induce a relatively robust humoral response. however, for n4ct1-ebovgp6, the ebovgp-specific humoral response does decline markedly, presumably as a result of the calculated 85% reduction in ebovgp expression when in the sixth position in the genome. previous work demonstrated that the n4ct1 vector backbone was highly attenuated in the mouse intracranial nv model [28, 30] . however, western blot data demonstrated that ebovgp was present on the surface of virus particles when expressed by an rvsv vector that retained the vsv g gene (unpublished data). therefore, a mouse nv study was performed to assess any changes in virulence arising from the possible alteration of vector tropism mediated by ebovgp. results confirmed the highly attenuated nature of n4ct1-ebovgp1 in the mouse central nervous system, indicating that the presence of ebovgp on the virion surface, in addition to vsv g protein, did not enhance virus spread in the brain, presumably because cells in the central nervous system lack the ebovgpspecific receptor [45] [46] [47] and because virus using vsv g receptors remained attenuated as previously described [30] . because n4ct1-ebovgp1 elicited a desirable balance of humoral and cellular immune responses to ebovgp after a single vaccine dose in mice, it was selected for guinea pig and nhp challenge studies. one dose of this vector protected guinea pigs from signs of disease following challenge with a lethal dose of gpa-ebov. interestingly, there was a slight delay in onset of the first signs of disease after challenge of wtvsv-vaccinated guinea pigs. one possible explanation is that induction of ifns and other innate immune responses by rvsv [48, 49] leaves the guinea pig innate immune system on high alert, even 21 days after vaccination, resulting in a generalized antiviral state that may initially slow the gpa-ebov infection [49, 50] . a single dose of n4ct1-ebovgp1 vector also protected nhps from disease following ebov challenge. an ebovgpspecific igg titer was detected in both immunized macaques prior to challenge and was expanded following ebov challenge, indicating that a preexisting ebovgp-specific antibody response is important for protection. unfortunately cell-mediated immune responses in nhps were not measured and may also contribute significantly to protection. the n4ct1-ebovgp1 vector used in these challenge studies is similar to the highly attenuated n4ct1-gag1 vector that demonstrated safety and immunogenicity in 2 phase 1 clinical studies. the studies described here are the first to demonstrate protection of guinea pigs and macaques with a single dose of highly attenuated rvsv expressing ebovgp, and we believe that the n4ct1-ebovgp1 vector has the essential safety and efficacy characteristics for use in a vaccine to prevent ebov infection in humans and the great apes. this highly attenuated vector platform will likely also have the potential to serve in a vaccine against other ebov strains that cause disease in humans and apes. ultimately, a multivalent ebov vaccine may be possible by combining ≥2 vaccine vectors in a single vaccine formulation. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations serologic cross-reactivity of human igm and igg antibodies to five species of ebola virus fruit bats as reservoirs of ebola virus spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species ebola-a growing threat? case fatality rate for ebola virus disease in west africa structure of the ebola virus glycoprotein bound to an antibody from a human survivor protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection progress in filovirus vaccine development: evaluating the potential for clinical use single immunization with a monovalent vesicular stomatitis virus-based vaccine protects nonhuman primates against heterologous challenge with bundibugyo ebolavirus recombinant vesicular stomatitis virusbased vaccines against ebola and marburg virus infections the epizootiology of the vesicular stomatitis viruses: a reappraisal vesicular stomatitis virus, indiana serotype: multiplication in and transmission by experimentally infected phlebotomine sandflies (lutzomyia trapidoi) vesicular stomatitis virus (indiana serotype): transovarial transmission by phlebotomine sandflies clinical and serological response to laboratory-acquired human infection by indiana type vesicular stomatitis virus (vsv) natural infection of humans, animals, and phlebotomine sand flies with the alagoas serotype of vesicular stomatitis virus in colombia recombinant vesicular stomatitis viruses from dna replicationcompetent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (rsv) antigens protect mice against rsv challenge long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine attenuated vesicular stomatitis viruses as vaccine vectors an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants sequential transcription of the genes of vesicular stomatitis virus order of transcription of genes of vesicular stomatitis virus phenotypic consequences of rearranging the p, m, and g genes of vesicular stomatitis virus moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates attenuation of recombinant vesicular stomatitis virus hiv-1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice in vivo biodistribution of a highly attenuated recombinant vesicular stomatitis virus expressing hiv-1 gag following intramuscular, intranasal, or intravenous inoculation synergistic attenuation of vesicular stomatitis virus by combination of specific g gene truncations and n gene translocations guide for the care and use of laboratory animals an efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development a simple method of estimating fifty percent endpoints pathogenesis of experimental ebola virus infection in guinea pigs modifying the hiv-1 env gp160 gene to improve pdna vaccine-elicited cell-mediated immune responses enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies vesicular stomatitis virus-based ebola vaccines with improved cross-protective efficacy vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with ebola and marburg viruses live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses structure-function analysis of the soluble glycoprotein, sgp, of ebola virus recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis ebola virus-like particles protect from lethal ebola virus infection impact of ebola mucin-like domain on antiglycoprotein antibody responses induced by ebola virus-like particles influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus gp dna vaccines ebola virus entry requires the cholesterol transporter niemann-pick c1 characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines distinct cellular interactions of secreted and transmembrane ebola virus glycoproteins transcriptional activation of alpha/beta interferon genes: interference by nonsegmented negative-strand rna viruses antiviral defense in mice lacking both alpha/beta and gamma interferon receptors natural killer cells in antiviral defense: function and regulation by innate cytokines financial support. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-000079-533xlisc authors: huszthy, peter c.; giroglou, tsanan; tsinkalovsky, oleg; euskirchen, philipp; skaftnesmo, kai ove; bjerkvig, rolf; von laer, dorothee; miletic, hrvoje title: remission of invasive, cancer stem-like glioblastoma xenografts using lentiviral vector-mediated suicide gene therapy date: 2009-07-20 journal: plos one doi: 10.1371/journal.pone.0006314 sha: doc_id: 79 cord_uid: 533xlisc background: glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. the translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model. methodology/principal findings: we used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. both, lymphocytic choriomeningitis virus glycoprotein (lcmv-gp) and vesicular stomatitis virus glycoprotein (vsv-g) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. in contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their cd133-, nestinand sox2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. in a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (hsv-1-tk) fused to egfp, both lentiviral vectors mediated a complete remission of solid tumors as seen on mri resulting in a highly significant survival benefit (p<0.001) compared to control groups. in all recurrent tumors, surviving egfp-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. conclusions/significance: in conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. the inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for gbm and thus confirms the high reproducibility of the invasive glioma animal model for translational research. glioblastoma is the most frequent and most malignant primary brain tumor. despite advances in neurosurgery, radiation and chemotherapy, the prognosis of patients remains poor with a median survival of 14 months [1] . a major drawback in translational brain cancer research has been the lack of suitable animal models. syngeneic-or xenograft tumors based on glioblastoma cell lines cultured as monolayers grow as circumscribed and highly angiogenic lesions in vivo [2] , lacking the invasive tumor cells, which represent an important feature of human glioblastoma. the invasive cells migrate away from the initial tumor mass and can cause recurrent tumors in different regions of the brain. thus, these cells represent a major therapeutic target. a recently established model in which glioblastoma biopsybased spheroids are serially passaged in the brains of nude rats shows highly invasive and angiogenic features [3] . therefore, this model is well suited for the study of new therapeutic strategies. still, reports using this or other clinically relevant models for experimental therapy are scarce. recently, we analyzed the therapeutic potential of the hsv-1-based oncolytic herpes vector g207 in the biopsy spheroid-based gbm model. the tumor volume in treated animals was reduced compared to control groups, but there was no significant survival advantage [4] . in contrast, the same therapy was more effective in a cell line-based animal model [5] and as a result is currently investigated in a phase i/ii clinical study [6] . in the present investigation we used the invasive xenograft model to evaluate transduction and therapeutic efficacy of lentiviral pseudotyped vectors. gammaretroviral vectors derived from the moloney murine leukemia virus (mmlv) have been the most frequently used retroviruses for gene therapy of brain tumors [7] [8] [9] [10] . however, despite promising results in animal models, clinical trials using retroviral vector supernatants or retroviral packaging cells have failed [11] [12] [13] . one major drawback of gammaretroviral vectors is the exclusive transduction of dividing cells, since in human gliomas, the majority of tumor cells do not divide within a given treatment window. therefore, lentiviral vectors with their ability to also transduce non-dividing cells are attractive candidates for the treatment of brain cancer. in previous studies, we have developed gammaretroviral and lentiviral vectors pseudotyped with the glycoproteins (gp) of the lymphocytic choriomeningitis virus (lcmv) [14, 15] . these vectors have a broad host range and can be concentrated by ultracentrifugation for in vivo applications. in addition, lcmv-gp is not cytotoxic, and stable recombinant packaging cell lines can be established [16, 17] . recently, we showed that lentiviral lcmv-gp pseudotypes efficiently delivered transgenes to rat glioma cells in vivo, while resident neurons were not transduced [18] . furthermore, we showed a significant therapeutic effect of lcmv-gp pseudotyped lentiviral vectors in the cell-line based 9l rat glioma model using the suicide gene hsv-1-tk. vsv-g lentiviral pseudotypes also showed a significant efficacy, similar to that of lcmv pseudotypes, which was mainly mediated by a bystander effect of transduced normal brain cells [19] . in the presented work, we showed that both, vsv-g and lcmv-gp pseudotyped lentiviruses efficiently transduced human glioma cells in vitro and in vivo, whereas gammaretroviral transduction was inefficient. the gene transfer to glioma cells was efficient for both lentiviral pseudotyped vector types. however, it was more specific using lcmv-gp pseudotyped vectors, as vsv-g pseudotypes also transduced host brain cells in invasive areas. analysis of transduced tumor cells revealed that both lentiviral vectors targeted cd133-positive as well as cd133negative cancer cells. furthermore, transduced glioblastoma cells expressed the stem cell markers nestin and sox2. importantly, when evaluated for therapeutic application using hsv-1-tk as a transgene, both lentiviral vectors mediated complete tumor regression on mri, resulting in a highly significant survival benefit (p,0.001) compared to the control groups. the collection of human biopsy tissue was approved by the regional ethical committee. the handling of the animals and the surgical procedures were done in accordance with the norwegian animal act and the local ethical committee approved the protocol. the human embryonic kidney cell line 293t (atcc number crl-11268) and the te671 cell line were obtained from the american type culture collection (atcc, manassas, va) and maintained in dulbbeco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs) and 1% glutamine. all cell lines were grown at 37uc in a humidified atmosphere of 5% co 2 . tumor fragments from glioblastoma multiforme patients were obtained during surgery. tissue specimens were taken from viable tumor areas that corresponded to regions with contrast enhancement on preoperative mri-scans. the specimens were transferred to test tubes containing complete growth medium, and spheroids were prepared as previously described [20] . the same method was applied for tumor material passaged in nude rats. briefly, tissue samples were minced into ,0.5 mm fragments and placed into 80 cm 2 tissue culture flasks (nunc, roskilde, denmark) base-coated with 0.75% agar (difco, detroit, mi). the spheroids were maintained in a standard tissue culture incubator with 5% co 2 and 100% relative humidity at 37uc. the medium was changed once a week. spheroids with diameters between 400 and 600 mm were selected for in vitro experiments and for intracerebral implantation. tumors were dissociated using the neuronal dissociation kit (miltenyi, bergisch-gladbach, germany) according to the manufacturer's protocol. cells were analyzed and sorted on a moflo cell sorter (beckman coulter, usa; former cytomation, usa), equipped with a coherent enterprise 621 argon-ion laser tuned to 488 nm (used at 180 mw), and 635 nm diode (25 mw). two mg/ml propidium iodide -pi (molecular probes) were added to the samples before flow sorting to facilitate dead cell discrimination. the gfp and pi were excited at 488 nm and fluorescence was measured through 530/40 bp and 613/20 bp optical filters (all filters from omega optical, brattleboro, vt, usa), respectively. doublets were discriminated using a forward light scatter (fsc) versus pulse width. fl3 channel (in logarithmic mode) with fsc were used to display and gate out pi positive/dead cells. fsc and side light scatter (ssc) signals were detected and shown in linear mode. gfp+ cells were defined on ssc versus fl1 (in logarithmic mode) dot plot after exclusion of dead cells and debris as described above. for analysis of cd133 expression cells were stained with allophycocyanin (apc) conjugated monoclonal cd133/1 (ac133) antibodies (miltenyi, bergisch-gladbach, germany), according to the manufacturer's general protocol for immunofluorescent staining (for 10 min in the dark at 4uc). cd133-apc was excited at 635 nm, the fluorescence was measured through 670/ 30 bp optical filter, and alive cd133+ cells were defined on ssc versus fl6 (in logarithmic mode) dot plot. non-stained cell suspension was used as a control. gfp+ tumor cells were sorted in ''purify 1'' mode into polypropylene round-bottom falcon tubes (becton dickinson labware europe, france) containing culture media, that were placed on ice. aliquots from some samples at the end of the sorting were removed and reanalyzed for control of the sort purity that was greater than 98%. sorted cells were either cultured in neurobasal medium (invitrogen, carlsbad, ca) with b27 supplement (20 ml/ml; invitrogen), glutamax (10 ml/ml; invitrogen), fibroblast growth factor 2 (20 ng/ml; peprotech, rocky hill, nj), epidermal growth factor (20 ng ml; peprotech) or transferred to dmem supplemented with 10% fetal calf serum (fcs) and 1% glutamine and grown on cover slips in 24 well plates. immunofluorescence staining of spheroids/adherent cells spheroids/adherent cells were stained with human-specific mouse-anti-nestin antibodies (millipore, billerica, ma), goat-anti-sox2 antibodies (r&d), mouse-anti-b2tubuliniii (millipore) antibodies and mouse-anti-gfap antibodies (millipore). primary antibodies were incubated overnight at 4uc. alexa-fluor647-goatanti-mouse und alexa-fluor647-donkey-anti-goat antibodies (dianova, hamburg, germany) were used as secondary antibodies over night at 4uc (for spheroids) or for 2h at room temperature (for adherent cells). spheroids were examined under a fluorescence microscope (nikon, tokyo, japan) and adherent cells were analyzed by confocal scanning laser microscopy (zeiss, jena, germany). the lentiviral vector plasmid prrl.sincmvegfppre was published by naldini et al. [21] . the construction of the lentiviral vector prrl.sincmv-tk/egfppre has been described previously. the retroviral vector pmp71-egfp-pre has been described previously [22] . the 293t cell line was used for transient lentiviral vector production. the lentiviral vector plasmid prrl.sincmv-tk/ egfppre (5 mg) or prrl.sincmvegfppre (5 mg), the hiv gagpol-rev expression plasmid pcmv-dr8.91(12.5 mg) [21] and 2 mg of the envelope expression plasmid phcmv-lcmv-gp [14] or pcmv-g [23] were cotransfected into 293t cells and concentrated as described previously [18] . for the production of retroviral vectors, 293t cells were transfected with 7.5 mg of pmp71-egfp-pre, 12.5 mg of psv-mo-mlvgagpol, and 2 mg of the envelope expression plasmid phcmv-lcmv-gp [14] or pcmv-g [23] . vectors were harvested and concentrated as described previously [24] . vectors were titered on te671 cells as described previously [18] . intracranial implantation of glioblastoma spheroids was done as described previously [25] . three weeks to one month after implantation, the animals were anesthetized and prepared for vector injection. the skin was withdrawn to reveal the location of the craniotomy. 2 times 10 ml of vector stocks were delivered into the centre of the tumors using a glass syringe (model 701, hamilton, bonaduz, switzerland) secured in a microprocessor-controlled infusion pump (ump 2-1, world precision instruments, aston, stevenage, uk). the injection coordinates were estimated after analyzing mri images for each individual lesion. vector infusion was done by convection enhanced delivery in the course of 25 min (200 nl/min for 10 min, followed by 400 nl/min for 10 min, and finally 800 nl/ min for 5 min). after infusion, the needle was left in place for 5 min to avoid vector reflux. the needle was slowly retracted and the skinfolds were closed with polyamide surgical thread. following surgery, rats were allowed to recover in an incubator set at 35uc before returning them to the cages. rats bearing glioblastoma xenografts were treated by daily i.p. injections of 50 mg/kg ganciclovir (gcv, roche, basel, switzerland). animals were euthanized and perfused with sterile saline and thereafter with 4% paraformaldehyde. brains were removed, suspended in 30% sucrose for three days, and then snap frozen in isopentane chilled with dry ice. coronal sections (12 mm) were prepared on a cryostat. for immunofluorescence analysis, sections were stained with human-specific anti-nestin antibodies (millipore) for human glioblastoma cells, mouse-anti-neun (millipore) antibodies for neurons, rat specific mouse-anti-nestin antibodies (millipore) for astrocytes and progenitor cells. primary antibodies (dilution 1:200) were incubated overnight at 4uc. biotinylated goat-anti-mouse and goat-anti-rabbit (vector laboratories, burlinghame, ca) were used as secondary antibodies (dilution 1:100) for 2 h at room temperature. sections were incubated with extravidin-cy3 (sigma, st. louis, mo) as fluorochrome (dilution 1:200) for 1 h at room temperature. the sections were examined under a fluorescence microscope (nikon) and analyzed by confocal scanning laser microscopy (zeiss, jena, germany). for analysis of transduction efficacy, consecutive sections (every 20.-30.) throughout the tumors were examined under a fluorescence microscope (nikon) with an automated stage using 106magnification. the transduction volume was calculated using nikon lucia imaging software. paraffin embedded formalin-fixed tissue sections from rat brain and patient material were placed in xylene bath for 263 minutes, absolute ethanol 263 minutes, 96% ethanol 262 minutes and finally in distilled water for 30 seconds for removal of paraffin and rehydration. epitope retrieval was performed by heating the sections at 99uc for 20 minutes in 10 mm citrate buffer at ph 6.0. the sections were incubated with a monoclonal human-specific anti-nestin antibody 1:200 in tbs/1%bsa over night at 4uc. a biotinylated goat-anti-mouse antibody (vector laboratories) was used as secondary antibody (dilution 1:100) for 1 h at room temperature followed by abc-complex incubation for 30 min. sections were developed with with 393-diaminobenzidine (dako cytomation), following the manufacturer's instructions. using a bruker pharmascan 7 tesla mr scanner (bruker biospin, billerica, ma), axial t2-weighted rare sequences were acquired (repetition time, 4,200 ms; echo time, 36 ms; slice thickness, 1 mm; field of view, 3.2 cm; matrix size, 2566256; 20 slices). during scanning, the animals were kept under anesthesia with 1.5% isofluorane (schering-plough, kenilworth, nj) mixed with 50% air and 50% o2. survival was analyzed by a log-rank test based on the kaplan-meier test using spss software. differences between pairs of groups were determined by the student's t-test. p values,0.05 were considered significant. human glioblastoma spheroids derived either directly from the patient (low generation) or from serial in vivo passages in the brains of nude rats (high generation), were infected with lentiviral lcmv-gp (5610 4 in 10 ml) or vsv-g pseudotyped lentiviral vectors (both 5610 4 particles in 10 ml) or with retroviral mlv-based vectors pseudotyped with lcmv-gp (1610 5 particles in 10 ml). the vectors were prepared in the same way for in vitro and in vivo experiments (see materials and methods). both lentiviral vectors transduced patient spheroids and high generation spheroids very efficiently ( figure 1 ). in contrast, retroviral vectors transduced only a few single cells in high generation spheroids ( figure 1 ) and failed to transduce patient spheroids (data not shown). in conclusion, both lentiviral vectors are much more efficient in transducing human glioblastoma spheroids in vitro than retroviral vectors. to compare the transduction efficiency of lentiviral and gammaretroviral vectors in vivo, we used a xenograft model that reflects the angiogenic and invasive features of human glioblastoma in situ. the xenograft also expresses the neural progenitor marker nestin and closely recapitulates the histology of the patient tumor ( figure 2 ). the vectors were injected into the center of progressively growing lesions using microprocessor-controlled stereotactic infusion. the injection coordinates were estimated after analyzing mri images for each individual lesion. the injection volume applied was 2610 ml and the vector titre 1610 7 / ml for all vectors. transduction efficiency was evaluated 7 days after vector injection. both lentiviral pseudotyped vectors showed very efficient transduction of the tumors ( figure 3a,d) . when analyzed at higher magnification, both lcmv-gp and vsv-g pseudotyped lentiviral vectors showed efficient transgene delivery to nestin-positive tumor cells in solid ( figure 3b ,e) and invasive tumor areas ( figure 3c ,f). in contrast, the retroviral vector only transduced a few scattered tumor cells near the injection site (figure 3 g,h) . for a quantitative comparison of transduction efficiency between the two lentiviral pseudotyped vectors, the gfp-positive areas were measured on histological slides (see material and methods). the total volume of transduced tumor tissue was 7.0563.51 mm 3 for lcmv-gp pseudotyped vectors and 4.0562.04 mm 3 for vsv-g pseudotyped vectors ( figure 3i) . although there was a difference in the mean, it was not statistically significant (p = 0.269) due to high interindividual differences (standard deviations). to analyze transduction specificity, histological sections of invasive tumor areas were stained for rat specific markers neun (for neurons) and nestin (for astrocytes and progenitor cells). lcmv-gp pseudotyped lentiviral vectors exclusively transduced tumor cells in all invasive areas ( figure 4a ), while normal brain cells were not transduced ( figure 4b,c) . also vsv-g pseudotyped vectors showed specific transduction of tumor cells in some invasive areas ( figure 4d,e) , however, they also transduced a few host brain cells at other sites ( figure 4g,h) . to analyze the potential of both lentiviral vectors to infect cancer stem-like cells, transduced tumors were enzymatically dissociated and cd133 expression was measured by flow cytometry. both vectors transduced cd133-positive and cd133-negative cells ( figure 5a ). although there were high interindividual differences in the fraction of total cd133-positive tumor cells in the different xenografts, both vectors showed similar efficiency in transducing cd133-positive cells, which was slightly higher than the overall fraction of cd133-positive cells (table 1) . the gfp-positive cells from tumors transduced with lcmv-gp or vsv-g pseudotyped lentiviral vectors were sorted and cultured in neural basal medium supplemented with egf and bfgf. transduced tumor cells from both vectors were able to form spheroids ( figure 5b,e) . spheroids expressed the stem cell markers nestin and sox2 ( figure 5c,d,f,g) . sorted cells were also plated under serum conditions. the cells continued to show significant expression of the stem cell markers nestin and sox2, but also of the differentiation markers gfap and b-tubuliniii ( figure 5h-o) . to evaluate the therapeutic efficacy of both lentiviral pseudotyoped vectors in the invasive xenograft model, vectors expressing the suicide gene hsv1-tk fused to egfp were injected into established tumors when visible on mri using the same method as described for the in vivo tropism study. the animals were treated daily with 50 mg/kg ganciclovir for 30 days starting 7 days post vector injection. tumor growth was measured every 7-14 days by mri. after 4 weeks of treatment, 7 out of 8 animals in both the lcmv-and the vsv-pseudotype treated groups had a complete remission on mri ( figure 6 ). one animal in each group had a stable disease until the end of gc treatment. all animals in the control groups developed large tumors during the treatment period of 30 days ( figure 6 ). both, lcmv-and vsv-pseudotype treated animals had a highly significant survival advantage (p,0.001) compared to the control groups ( figure 7a ). there was no statistically significant difference in survival between the two treatment groups. upon cessation of prodrug administration, all animals developed recurrent tumors, which could be classified into three different groups ( figure 7b-e, table 2) . the animals showed either 1) local recurrences or 2) local and/or contralateral recurrences or 3) recurrences in other distant brain areas. there was no clear difference in the recurrence pattern between the two vector types, but lcmv-pseudotyped vector-treated animals had more contralateral recurrences, whereas vsv-g pseudotyped vector-treated animals had more local recurrences ( figure 7b -e, table 2). histopathological and confocal microscopic analysis of the lesions revealed gfp-positive cells in all recurrent tumors demonstrating that not all transduced glioma cells were killed by ganciclovir treatment (figure 7f-m) . in vsv-g pseudotype-treated animals, the gfp-positive surviving cells were found in invasive areas ( figure 7f ,g), the corpus callosum region ( figure 7h ) and also in some regions of distant recurrences. one animal also showed a focus of transduced normal brain cells at the tumor border that survived gc treatment ( figure 7i ). in the lcmv group, most gfp-positive cells were found in the ipsilateral hemisphere, in solid and invasive tumor areas ( figure 7j ,k,l), with only a few cells seen in the contralateral hemisphere ( figure 7m ). future success of glioma gene therapy will depend on more potent vector systems that show higher transduction efficiency than the systems that are available today. in addition, the application of representative animal models that recapitulate both, the invasive and angiogenic features of patient tumors, is vital in order to minimize the huge discrepancies between the experimental results and clinical outcomes previously observed for gene therapeutic strategies for brain cancer. to this end, we applied one of the most clinically relevant animal models for glioblastoma known. this model was established several years ago [20] and its growth pattern as well as geno-and phenotypic similarity to glioblastoma in patients has been extensively characterized [3] . a striking difference of our model compared to other cell-line based models is the highly invasive behaviour of the lesions, similar to glioblastoma in patients. our model is based on spheroids derived from patient biopsies that are passaged serially in the brains of nude rats. first generation tumors are highly invasive and grow without signs of angiogenesis. late generation tumors show an angiogenic phenotype, but are still invasive. our in vitro experiments revealed that both lentiviral vectors transduced spheroids derived from both low and high generation tumors very efficiently. in contrast, retroviral vectors transduced only high generation spheroids and displayed a much lower efficiency of gene transfer than both lentiviral vectors. this difference can only be attributed to the vector backbone, as the glycoprotein which is responsible for virus entry into the cell was the same for the retroviral and one of the lentivral vectors applied (lcmv-gp). the most important feature that distinguishes lentiviral from retroviral vectors is their ability to infect non-dividing cells. it is known that glioma spheroids, especially primary biopsy spheroids, contain a significant fraction of non-dividing cells, which cannot be transduced by retroviral vectors. it has previously been shown that the cultured biopsy spheroids show a similar cell proliferation as seen in glioblastoma patients [20] . previous studies have demonstrated that retroviral vectors can very efficiently transduce highly proliferative monolayer cultures of glioma cell lines [14] . however, monolayer cultures change their geno-and phenotypic characteristics under long term culture and thus are not a suitable model to answer clinically relevant questions [26] . for in vivo experiments, we selected a high generation xenograft that showed both angiogenic and invasive features and recapitulated the histology of the patient lesion. furthermore, this xenograft showed a high level of nestin expression similar to the patient material. in translational research it is crucial to assure that the experimental tumors truly reflect the corresponding patient's tumor properties to avoid using non-relevant animal models. however, this strategy is not common practice yet, based on the simplicity of using established cell lines for in vivo experiments. we showed a high transduction efficiency of lentiviral vectors for glioma cells in vivo, whereas retroviral vectors transduced a few scattered tumor cells near the injection track. this is in contrast to in vivo studies by others where retroviral vectors were very efficient, but as mentioned above, the models applied were non-invasive, based on cell lines cultured as monolayers [27] . of note, the results of clinical studies using retroviral vectors showed the same low transduction efficiency as observed in our model system [12] . this finding provides further evidence that the glioma model used here has a higher predictive value for the performance of a novel therapeutic approach in the clinic than previous animal models. the tropism for glioma cells was more specific with lcmv-gp lentiviral pseudotyped vectors, as vsv-g pseudotyped lentiviral vectors also transduced few normal brain cells in invasive areas. in previous studies using a rat glioma model, we also showed a more specific transduction of glioma cells by lcmv-gp pseudotyped vectors compared to vsv-g pseudotyped vectors [18, 19] . in fact, in these studies, the vsv-g pseudotyped vectors transduced normal brain cells at a much higher frequency than in this study. this can be explained by the mode of vector delivery, because in the previous studies, we injected the vectors both into the tumor core as well as into tumor border areas. in the present study, we injected the vectors into the tumor core only using convection enhanced delivery. we used this method because it results in a high distribution volume of drug and vector and is currently used as a delivery method in clinical studies [28] . in addition, the tumor model we apply here is highly invasive and lacks a sharply demarcated brain tumor/normal brain interface, present in the rat glioma model. another explanation could be the species difference as we used human glioma cells in this study in contrast to rat glioma cells in the previous work. vsv-g pseudotyped vectors might have a higher tropism for human glioma cells than for rat normal host cells. however, as the receptor for vsv-g is unknown [29] , this remains a hypothesis. the targeting of cancer stem cells or cancer stem-like cells in human tumors including glioblastoma has recently evolved as a major aim in cancer therapy. these stem cells are suggested to initiate cancer and might be resistant to therapy, thus being responsible for tumor recurrence [30] [31] [32] . yet, recent studies have initiated a controversial discussion whether cancer stem cells really exist [33, 34] . therefore, we use the term ''cancer stem-like cells'' in our study to designate cells which have certain stem-like properties described previously. we showed that both lentiviral vectors transduced cd133-positive and cd133-negative cells. cd133-positive cells have been identified as cancer initiating cells and cancer stem cells in many different cancers including glioblastoma [30, 35] . however, more recent reports questioned these findings showing that cd133-negative popopulations can include cancer initiating cells as well [36, 37] . the efficient targeting of cd133-positive and cd133-negative glioma cells has also been described for adenoviral vectors [38, 39] . we further demonstrated that sorted cells from tumors transduced either by lentiviral lcmv-gp or vsv-g pseudotyped lentiviral vectors had the ability to form spheroids upon culturing in neural basal medium supplemented with egf and bfgf. spheroids from both sorted cell populations expressed the neural stem cell markers nestin and sox2 and showed the ability to express the differentiation markers gfap and b-tubuliniii under serum conditions. these properties have been described for neural progenitor cells [40] as well as for cancer stem-like cells in human glioblastoma [26] . thus, the cell populations transduced by lcmv or vsv pseudotyped lentiviral vectors showed features of cancer stem-like cells which might be an important target for therapy. in a therapeutic approach using the suicide gene hsv-1-tk fused to egfp, we demonstrated a highly significant therapeutic effect for both lentiviral vectors compared to control groups. using mri to follow tumor growth, we detected complete remission in 7 out of 8 animals for lcmv-gp and vsv-g pseudotyped vectors after 30 days of ganciclovir treatment. hsv-1-tk has been reported to be an effective therapeutic gene in previous studies [19, 41] . the limited success in clinical studies has been a result of inefficient gene delivery systems rather than lack of efficacy of the suicide mechanism [11] . however, there is still space for improvement of the prodrug delivery such as application length, treatment intervals and route of delivery. tai et al. demonstrated that multiple cycles of prodrug application are superior over one cycle of prodrug [42] . in our study, we still detected gfp-positive tumor cells after one cycle (30 days) of ganciclovir administration in treated animals indicating that application in cycles might also improve the therapeutic effect in this setting. further, these results demonstrate that vector-transduced tumor cells retain the ability to invade brain tissue and migrate even to distant brain regions. in conclusion, the present study demonstrates an efficient transduction and therapy of experimental human glioblastoma by lentiviral vectors. the inefficient gene transfer by gammaretroviral vectors is in line with the results obtained in clinical trials and thus confirms the high relevance of the spheroid-based glioma animal model for translational research. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma expression of extracellular matrix components in a highly infiltrative in vivo glioma model angiogenesis-independent tumor growth mediated by stem-like cancer cells oncolytic herpes simplex virus type-1 therapy in a highly infiltrative animal model of human glioblastoma attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas phase ib trial of mutant herpes simplex virus g207 inoculated pre-and post-tumor resection for recurrent gbm eradication of rat malignant gliomas by retroviral-mediated, in vivo delivery of the interleukin 4 gene inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2 eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene highly efficient and tumorrestricted gene transfer to malignant gliomas by replication-competent retroviral vectors clinical trials with retrovirus mediated gene therapywhat have we learned? a phase iii clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range retroviral vectors pseudotyped with lymphocytic choriomeningitis virus recombinant expression of lymphocytic choriomeningitis virus strain we glycoproteins: a single amino acid makes the difference a retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins normal brain cells contribute to the bystander effect in suicide gene therapy of malignant glioma multicellular tumor spheroids from human gliomas maintained in organ culture in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors a general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein widespread dispersion of adeno-associated virus serotype 1 and adenoassociated virus serotype 6 vectors in the rat central nervous system and in human glioblastoma multiforme xenografts tumor stem cells derived from glioblastomas cultured in bfgf and egf more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines vesicular stomatitis virus g pseudotyped retrovector mediates effective in vivo suicide gene delivery in experimental brain cancer convection enhanced drug delivery of novel therapeutic agents to malignant brain tumors phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus identification of human brain tumour initiating cells isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma glioma stem cells promote radioresistance by preferential activation of the dna damage response tumor growth need not be driven by rare cancer stem cells efficient tumour formation by single human melanoma cells identification of a cancer stem cell in human brain tumors cd133(+) and cd133(2) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles cd133 negative glioma cells form tumors in nude rats and give rise to cd133 positive cells lowdose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells adenoviruses 16 and cv23 efficiently transduce human low-passage brain tumor and cancer stem cells isolation and characterization of neural progenitor cells from post-mortem human cortex bystander killing of malignant glioma by bone marrow-derived tumorinfiltrating progenitor cells expressing a suicide gene single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma we thank mariana carstov, narve brekka, roswitha seyd, hege dale and endy spriet for expert technical assistance. key: cord-291323-kbjyd5g3 authors: kang, yuan-lin; chou, yi-ying; rothlauf, paul w.; liu, zhuoming; soh, timothy k.; cureton, david; case, james brett; chen, rita e.; diamond, michael s.; whelan, sean p. j.; kirchhausen, tom title: inhibition of pikfyve kinase prevents infection by zaire ebolavirus and sars-cov-2 date: 2020-08-25 journal: proc natl acad sci u s a doi: 10.1073/pnas.2007837117 sha: doc_id: 291323 cord_uid: kbjyd5g3 virus entry is a multistep process. it initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. we describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (vsv) containing the envelope proteins of zaire ebolavirus (vsv-zebov) or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (vsv-sars-cov-2) elicited by apilimod and vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, pikfyve. we also describe potent inhibition of sars-cov-2 strain 2019-ncov/usa-wa1/2020 by apilimod. these results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of pikfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against sars-cov-2. virus entry is a multistep process. it initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. we describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (vsv) containing the envelope proteins of zaire ebolavirus (vsv-zebov) or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (vsv-sars-cov-2) elicited by apilimod and vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, pikfyve. we also describe potent inhibition of sars-cov-2 strain 2019-ncov/usa-wa1/2020 by apilimod. these results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of pikfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against sars-cov-2. covid-19 | sars-cov-2 | zebov | apilimod | vacuolin-1 m embrane-enveloped viruses deliver their contents to cells via envelope protein-catalyzed membrane fusion. binding of virus to specific host cell receptor(s) triggers membrane fusion, which can occur directly at the plasma membrane or following endocytic uptake. viruses that require endocytic uptake can use different initial trafficking routes to reach the site of membrane fusion. in endosomes, acidic ph serves to trigger conformational rearrangements in the viral envelope proteins that catalyze membrane fusion, as seen for influenza a virus and vesicular stomatitis virus (vsv). for zaire ebolavirus (zebov), proteolytic processing of the envelope protein by host cell proteases (1) is necessary to expose the receptor binding domain prior to engagement of niemman−pick disease type 1c (npc1 or npc intracellular cholesterol transporter 1)-the late endosomal−lysosomal receptor protein (2) . proteolytic processing is also required for severe acute respiratory syndrome coronavirus (sars-cov) (3, 4) , and for the current pandemic sars-cov-2 (5). lassa fever virus (lasv) uses a different mechanism, binding alpha-dystroglycan at the plasma membrane (6) , for internalization with a subsequent ph-regulated switch that leads to engagement of lysosomalassociated membrane protein 1 for membrane fusion (7) . lymphocytic choriomeningitis virus (lcmv) also uses alpha-dystroglycan (6) and is internalized in a manner that depends on endosomal sorting complexes required for transport proteins (8) , although it remains unknown whether a second receptor is required. a hallmark of the endolysosomal system is controlled dynamic trafficking of vesicular carriers among its various subcompartments. phophoinositides are markers for defining the identity of these subcompartments, because they are restricted in their distribution to specific intracellular membranes (reviewed in ref. 9 ). although it is one of the least abundant of the phosphoinositides in cells, pi (3, 5) p2 is particularly important for endomembrane homeostasis. it is produced by pi-3p-5-kinase (pikfyve), which phosphorylates the d-5 position in phosphatidylinositol-3-phosphate (pi3p) to yield phosphatidylinositol 3,5-bisphosphate (pi(3,5)p2) (10) . first cloned as mammalian p235 (11) , pikfyve is a 240-kda class iii lipid kinase, present on the cytosolic face of endosomal membranes (12, 13) as part of a ternary complex with the pi(3,5)p2 5-phosphatase sac3 and arpikfyve (14) . ablation of pikfyve function by genetic (12, 15) or pharmacological means (16) (17) (18) (19) (20) causes endosomal swelling and vacuolation of late endosomes and endolysosomes. it is thought that these changes result from decreased membrane fission and concomitant interference in endosomal traffic (13, 21) . small-molecule inhibitors of pikfyve, all of which have some structural resemblance to each other, have been studied as potential drugs for treating cancer and autoimmune diseases. these inhibitors include apilimod (19) , vacuolin-1 (18), a series of 30 vacuolin-related the membrane fusion proteins of viral pathogens as diverse in their replication strategies as coronaviruses and filoviruses depend, for their functional activity, on proteolytic processing during cell entry. endosomal cathepsins carry out the cleavages. we have constructed chimeric forms of vesicular stomatitis virus (vsv) bearing the fusion proteins of zaire ebolavirus (zebov) or sars coronavirus 2 (sars-cov-2) and shown that two small-molecule inhibitors of an endosomal lipid kinase (pikfyve) inhibit viral infection by preventing release of the viral contents from endosomes. both inhibitory compounds cause distension of rab5 and rab7 subcompartments into small vacuoles. one of them (apilimod) also inhibits infection of cells by authentic sars-cov-2. the results point to possibilities for host targets of antiviral drugs. molecules (22) , ym201636 (16) , and wx8 chemical family members (20) . physiological effects of these compounds in cells include inhibition of autophagy (17, 22, 23) , reduced generation of il-12/ il-23 (24) , and reduced dendritic cell infiltration in psoriasis (25) . apilimod also inhibits infection by several viruses, including zebov. although it does not alter the ph of endosomes nor inhibit cathepsin b or l (26), apilimod blocks entry of zebov and other pathogenic filoviruses (27) . several groups reported that apilimod prevents colocalization of vsv-zebov pseudoviruses with the zebov endosomal receptor npc1, but does not prevent colocalization with early endosomal antigen 1 (eea1) (5, 27, 28) . apilimod also inhibits entry of pseudotyped viruses bearing the spike proteins of middle east respiratory syndrome cov, sars-cov, and sars-cov-2, as well as of authentic mouse hepatitis virus particles (5) . here, we have studied the effects of apilimod on infection of vsv-egfp-sars-cov-2 and vsv-egfp-zebov chimeras and showed that apilimod blocks infection of both, with a concentration that inhibits response by 50% (ic 50 ) of ∼50 nm. apilimod and vacuolin-1 also prevented entry and infection of vsv-megfp-zebov and many of the internalized vsv-megfp-zebov virions colocalized with npc1 in the distended, vacuolated endosomes. this suggests that blocking pikfyve kinase has the same downstream effects on these viruses, even though vsv-egfp-sars-cov-2 does not require interaction with npc1 for membrane fusion. apilimod also inhibits infection by authentic sars-cov-2 strain 2019-ncov/usa-wa1/2020 virus, with an ic 50 slightly lower than the ic 50 for the vsv-egfp-sars-cov-2. we suggest that apilimod, which has passed safety tests in previous human clinical trials for nonviral indications (24, 25, 29, 30) , is a potential starting point for developing small-molecule entry inhibitors of sars-cov-2 that could limit infection and disease pathogenesis. apilimod inhibits infection of vsv-megfp-lcmv and vsv-zebov. we inoculated svg-a cells with vsv chimeras expressing the viral matrix protein (m) fused to egfp (megfp). the chimeras include vsv (vsv-megfp, which initiates fusion at ph < 6.2), vsv-v269h gp (vsv-megfp-v269h, a variant of vsv gp that initiates fusion at ph < 5. potently inhibit vsv-megfp-zebov infection (fig. 1c) . these results agree with results obtained by others with apilimod (26, 31) in different cell types infected with murine leukemia virus (mlv) virus pseudotyped with zebov gp or with ebola virus itself (26, 27, 32) . apilimod was a less effective inhibitor of vsv-megfp-lcmv infection, and vacuolin-1 had no effect at the concentration used. in contrast, apilimod and vacuolin-1 failed to prevent infection by vsv-megfp, vsv-megfp-v269h, vsv-megfp-rabv, or vsv-megfp-lasv (fig. 1c) . in1 (33) , an inhibitor of the phosphoinositide kinase vps34, the main endosomal generator of pi3p, also interfered with vsv-megfp-lcmv and vsv-megfp-zebov infection (fig. 1c ). all of these viruses require low ph to trigger viral membrane fusion with the endosomal membranes, and, as expected, infection was fully blocked by bafilomycin a1, which inhibits the vacuolar type h + -atpase (v-atpase) acidification activity (fig. 1c ). experiments, we deemed rnp delivery, as monitored by single-cell fluorescence microscopy imaging (experimental protocol summarized in figs expected, bafilomycin a1 blocked entry of all viruses (images in fig. 2c and quantification in fig. 2d ). u18666a, a potent inhibitor of npc1 (fig. 4f) , showed that npc1-halo remained active as a cholesterol transporter. using live-cell spinning disk confocal microscopy ( figs. 3 and 4) , we monitored the presence of virus particles in the fluorescently tagged endosomes by colocalization with the fluorescent spots from the virus-incorporated megfp. we monitored entry by carrying out the experiments in the presence of cycloheximide, thus ensuring that any megfp fluorescent signal at the nuclear margin originated only from megfp molecules carried by incoming viral particles (fig. 3 b and f) . all cells were maintained at 37°c throughout all phases of the experiment to ensure normal and undisturbed intracellular trafficking. all control experiments performed in the absence of inhibitors showed arrival of vsv-megfp, vsv-megfp-v269h, or vsv-megfp-zebov virus particles to early (rab5c and eea1) (figs. 3e and 4e) or late (rab7a or npc1) endosomes and lysosomes (figs. 3i and 4 c and e). megfp released from all viruses appeared at the nuclear margin, showing effective rnp release. npc1, the receptor for vsv-megfp-zebov entry, is required for fusion from endosomes (2) . the successful vsv-megfp-zebov infection observed in the absence of drug in cells expressing npc1-halo alone or in combination with mscarlet-eea1 indicates that npc1-halo is capable of facilitating infection and that vsv-megfp-zebov trafficked to npc1-halo−containing endosomes. apilimod and vacuolin-1 treatment of the svg-a cells led to enlargement and vacuolization of their endosomes and lysosomes tagged with fluorescent eea1, rab5c, rab7a, or npc1 (figs. [3] [4] [5] , in agreement with earlier pikfyve ablation studies (13, 21) . vsv-megfp and vsv-megfp-v269h (fluorescent dots, white) reached all tagged species of enlarged endolysosomes and successfully penetrated into the cytosol, as indicated by megfp at the nuclear margin (fig. 3 e and i) . vsv-megfp-zebov also trafficked to all tagged species of enlarged endolysosomes (fig. 3 e and i) , often reaching one of the numerous npc1-containing vacuoles enriched in eea1 (figs. 4e and 5 b and c). vsv-megfp-zebov in eea1-containing endosomes increased in the presence of apilimod, as also reported for vlp zebov (27) . while able to reach npc1containing functional endosomes in cells treated with apilimod apilimod blocks infection of vsv sars-cov-2. using a recombinant vsv expressing soluble egfp (vsv-egfp) where the glycoprotein (gp) was replaced with that of zebov gp (vsv-egfp-zebov) or sars-cov-2 s (vsv-egfp-sars-cov2), we inoculated ma104 cells with these chimera viruses and tested the effects of apilimod on infection by flow cytometry (fig. 6a) . we found potent inhibition of vsv-egfp-sars-cov-2 infection by apilimod and confirmed that the compound also inhibits vsv-egfp-zebov infection (fig. 6b) . the dose-response curves indicated similar effects for vsv-egfp-zebov and vsv-egfp-sars-cov-2 (ic 50 s of ∼50 nm), in contrast to the absence of any detectable inhibition of vsv-egfp infection, used here as a negative control. apilimod blocks infection of sars-cov-2 virus. to test the effect of apilimod on bona fide sars-cov-2 infection, we exposed vero e6 cells to fully infectious sars-cov-2 (strain 2019-ncov/ usa-wa1/2020); after 24-h incubation, supernatants were harvested and titered by focus-forming assay on a separate set of vero e6 cells (fig. 7a) . apilimod strongly inhibited sars-cov-2 infection, and the dose-response curve was similar or more potent than those observed for vsv-egfp-zebov or vsv-egfp-sars-cov-2 (ic 50 s of ∼10 nm) (fig. 7b ). coronaviruses, filoviruses, and arenaviruses have different replication strategies and unrelated surface gps that engage different receptor molecules during entry (1, 2, 5-8). coronavirus and filovirus surface gps share a requirement for entry-associated proteolytic processing for activation as fusogens (1). filoviruses require passage through low-ph compartments where cathepsins are active. coronaviruses may enter directly by fusion at the plasma membrane or following receptor-mediated endocytosis. cell entry of sars-cov and sars-cov-2 depends on the protease tmprss2 in conjunction with ace2 (34) (35) (36) (37) , and, when tmprss2 is present, the entry pathway becomes insensitive to cathepsin inhibition (34, 37, 38) . the common inhibition of viruses from all three groups by apilimod is a consequence of perturbing their shared entry pathway. moreover, it is not the cathepsin activity itself that these compounds affect, judging from the outcome of the assays with apilimod and vacuolin-1 showing they inhibit vsv chimeras bearing the surface gps of zebov and lcmv and, to a lesser extent, lasv. apilimod also inhibits infection of cells by vsv-sars-cov-2 as well as by authentic sars-cov-2; neither compound blocks infection by wild-type vsv. for vsv-zebov, we have shown that the virus reaches a compartment enriched in npc1, the zebov coreceptor, and often also enriched in eea1, but that it nonetheless fails to release internal proteins into the cytosol. apilimod does not inhibit cathepsin (26) , but apilimod (39) and vacuolin-1 (17, 23) can interfere with cathepsin maturation, as evidenced by an increase in procathepsin in treated cells; they do not influence endosomal ph (18, 26, 40) , although other studies report that apilimod decreases cathepsin activity (41) and vacuolin-1 increases ph (17, 23) . irrespective of this discrepancy, both apilimod and vacuolin-1 inhibit pikfyve (17, 19) , a three-subunit complex (14) with a pi-3p−binding fyve domain (10, 11) that recognizes the endosomal marker, pi-3-p. functional ablation of this enzyme by genetic means (12, 15) gives rise to the same cellular phenotype as treatment with either compound (17) (18) (19) . the similar dose-response curves for apilimod inhibition of the zebov and sars-cov-2 chimeras (ic 50 of ∼50 nm) and of authentic sars-cov-2 virus (ic 50 of ∼10 nm) are in good agreement with the ic 50 of ∼15 nm for apilimod inhibition of pikfyve in vitro (19) . thus, perturbing normal endosomal trafficking by inhibiting pikfyve activity suggests it is the mechanism by which apilimod and vacuolin-1 block entry of such a diverse set of viral pathogens. one of the most striking consequence of pikfyve inhibition, and hence of pi-3,5-p 2 restriction in endosomal membranes, is the swelling of endosomes into small, spherical vacuoles-the phenomenon that gave vacuolin-1 its name (18) . our imaging data with vsv-megfp-zebov chimeras show that the virus particles accumulating in these structures, many of which also contain the npc1 coreceptor (2, 42) , often appear to be relatively immobile and adjacent to the endosomal limiting membrane. one possible explanation is that, when a virion reaches these distended endosomes, it can bind or remain bound to the limiting membrane, but not fuse. another is that virions may fuse with smaller intraluminal vesicles in the endosomal lumen (43) , but that pi-3,5-p2 depletion prevents back-fusion of these vesicles with the endosomal limiting membrane and inhibits release into the cytosol of the viral genome. inhibition of infection by authentic sars-cov-2 shows that the blocked release of the viral genome from a vacuolated endosome is independent of the shape, size, and distribution of spike protein on the virion. the assay we used to determine effects on infectivity of authentic virus measured release of virions after multiple rounds of infection, rather than entry, which we monitored in the vsv-sars-cov-2 experiments by detecting egfp synthesis in the cytosol. nevertheless, the ic 50 of apilimod in experiments with authentic virus is remarkably similar to (or even more potent than) that obtained with chimeric vsv-sars-cov-2. although cathepsin l inhibitors block sars-cov and sars-cov-2 infection in cell culture (4, 5) , they have less pronounced effects when tested in animals (44) . this may because another protease, tmprss2 on the surface of cells in relevant tissues, appears to prime sars-cov (44) and sars-cov-2 (37) spike proteins for efficient entry. as the effectiveness of apilimod and vacuolin-1 does not depend on cathepsin inhibition, their capacity to block entry of several distinct families of viruses is likely to be independent and downstream of the protease that primes their surface gp for fusion. phase i and phase ii clinical trials have shown that apilimod is safe and well tolerated (24, 25, 29, 30) . the trials were discontinued because of lack of effectiveness against the autoimmune condition for which the drug was tested. we suggest that one of these compounds, or a potential derivative, could be a candidate broad-spectrum therapeutic for several emerging human viral pathogens, including sars-cov-2. cell culture. human astroglial svg-a derived cells (a kind gift from walter j. atwood, brown university, providence, ri) were grown at 37°c and 5% co 2 in minimum essential medium (mem) (10-010-cv; corning) supplemented with 10% heat-inactivated fetal bovine serum (fbs, s11150; atlanta biologicals), penicillin, and streptomycin (1406-05-9; vwr international). sars-cov-2 strain 2019-ncov/usa-wa1/2020 was obtained from the centers for disease control and prevention (gift of natalie thornburg, centers for disease control, atlanta, ga). virus was passaged once in vero ccl81 cells (atcc) and titrated by focus-forming assay also on vero e6 cells. genome editing. individual cell lines of svg-a were gene edited in both alleles using the crispr-cas9 system to incorporate fluorescent tags into the n terminus of rab5c (tagrfp), rab7a (tagrfp), eea1 (mscarlet), or the c terminus of npc1 (halo). the npc1-halo expressing cells were further gene edited to incorporate mscarlet-eea1 creating svg-a cells simultaneously expressing mscarlet-eea1 and npc1-halo. a free pcr strategy (49, 50) was used to generate small guide rnas (sgrna) with target sequences for either rab5c, rab7a, npc1, or eea1 (table 1) . the genomic dna fragment of rab5c, rab7a, npc1, or eea1 genes fused with either tagrfp, halo, or mscarlet were cloned into the puc19 vector (donor constructs) which then served as homologous recombination repair templates for the cas9 enzyme-cleaved genomic dna. donor constructs were obtained by a ligation of pcr amplification products from the genomic dna fragments, tagrfp, halo, and mscarlet sequences. primers f1-r1 and f3-r3 amplified ∼800 base pairs of genomic sequences upstream and downstream of the start codon of rab5c, rab7a, or eea1 or the stop codon of npc1, respectively (table 1) . primers f1 and r3 contain sequences complementary to the puc19 vector linearized using the smai restriction enzyme (lowercase in the primer sequences). the tagrfp sequence containing the ggs peptide linker was amplified using primers f2-r2 from a tagrfp mammalian expression plasmid used as a template. the f2 primer contains complementary sequences to the 3′ end of the f1-r1 fragment, while the f3 primer contains complementary sequences to the 3′ end of the tagrfp sequences. pcr products (fragments f1-r1, f2-r2, and f3-r3) were subjected to electrophoresis in 1% agarose and gel purified using a purification kit from zymogen. the pcr fragments were cloned into the linearized puc19 vector using the gibson assembly cloning kit (e5510s; new england biolabs). svg-a cells (1.5 × 10 5 cells) were cotransfected with 0.8 μg of streptococcus pyogenes cas9, 0.8 μg of free pcr product coding for the target sgrna, and 0.8 μg of puc19 vector using lipofectamine 2000 reagent (invitrogen) according to the manufacturer's instructions. transfected cells were grown for 7 d to 10 d and sorted for tagrfp, halo, or mscarlet expression using fluorescence-activated cell sorting (facs) (sh-800s; sony). prior to facs, npc1-halo cells were labeled for 15 min with janelia fluor 647 (jf647). single cells expressing the desired chimera were isolated, clonally expanded, and then screened by genomic pcr for tagrfp, halo, or mscarlet insertion into both alleles (primers listed in table 2 ). infection assays. svg-a cells were plated at about 30 to 40% confluency into 24-well plates and incubated for 1 d at 37°c and 5% co 2 . at the start of the experiment, cells were incubated with the indicated drug or dimethyl sulfoxide (dmso) at 37°c for 1 h. following this, cells were incubated for 1 h at 37°c with vsv, vsv-megfp-v269h, vsv-megfp-rabv, vsv-megfp-lasv, vsv-megfp-lcmv, or vsv-megfp-zebov in drug-or dmso-containing infection medium (α-mem, 50 mm hepes, 2% fbs). cells were then washed to remove nonadsorbed viruses and further incubated at 37°c in medium containing the drug or dmso, with experiments ending at the indicated times by fixation with 3.7% formaldehyde in phosphate-buffered saline (pbs). fluorescent intensity from 20,000 single cells from a single round of the sgrnas with target sequences specific for rab5c, rab7a, npc1, or eea1 were generated using a free pcr strategy using a u6 promoter-containing primer and reverse primers including rab5c, rab7a, npc1, or eea1 specific target nucleotide sequences (at the positions indicated with n). infection was determined by flow cytometry using a bd facscanto ii equipped with diva software package. ma104 cells were pretreated for 1 h with the indicated concentration of apilimod or dmso. pretreated cells were inoculated with vsv-egfp, vsv-egfp-zebov, or vsv-egfp-sars-cov-2 at a multiplicity of infection (moi) = 1 (based on titers in ma104 cells) in the presence of apilimod or dmso for 1 h at 37°c. at 6 to 8 h postinfection, cells were collected and fixed in 2% paraformaldehyde (pfa) and then subjected to flow cytometry. the percentage of gfp cells was determined using flowjo software (tree star industries). vero e6 cell monolayers were pretreated for 1 h at 37°c with serial dilutions of apilimod at the indicated concentrations. next, sars-cov-2 was diluted to an moi of 0.01 focus-forming units per cell in apilimod-containing medium and added to vero e6 cells for 1 h at 37°c. after adsorption, cells were washed once with pbs, and medium containing the respective concentration of apilimod was added. cells were incubated for 24 h at 37°c, at which time cell culture supernatants were removed and used for determination of viral titer by focus forming assay. sars-cov-2 focus-forming assay. cell culture supernatants from virusinfected cells were diluted serially 10-fold, added to vero e6 cell monolayers in 96-well plates, and incubated at 37°c for 1 h. subsequently, cells were overlaid with 1% (wt/vol) methylcellulose in mem supplemented with 2% fbs. plates were harvested 30 h later by removing overlays and fixed with 4% paraformaldehdye in pbs for 20 min at room temperature. plates were washed and sequentially incubated with 1 μg/ml cr3022 anti-spike antibody (51) and hrp-conjugated goat anti-human igg in pbs supplemented with 0.1% saponin and 0.1% bovine serum albumin (bsa). sars-cov-2−infected cell foci were visualized using trueblue peroxidase substrate (kpl) and quantitated on an immunospot microanalyzer (cellular technologies). data were processed using prism software (graphpad prism 8.0), and viral titers are reported as percent inhibition relative to mocktreated sars-cov-2−infected cells. entry assay and intracellular traffic. svg-a cells plated on glass #1.5 coverslips at about 30 to 40% confluency 1 d prior to experiment were treated with drug or dmso for 1 h at 37°c. following this, cells were incubated at 37°c with vsv, vsv-megfp-v269h, vsv-megfp-rabv, vsv-megfp-lasv, vsv-megfp-lcmv, or vsv-megfp-zebov in drug-or dmso-containing infection medium. after this, cells were washed, then further incubated in medium containing the drug or dmso at 37°c, with the experiment ending at the indicated time by fixation for 20 min at room temperature with 3.7% formaldehyde in pbs. this was followed with a 10-min incubation of 5 μg/ml alexa647-labeled wheat germ agglutinin in pbs to label the outline of the cells. cells were imaged using a spinning disk confocal microscope with optical planes spaced 0.3 μm apart (52) . the entry assay scored the presence of megfp at the nuclear margin in each cell. trafficking of viruses to endosomal compartments was observed using live-cell imaging using the spinning disk confocal microscope. chemical fixation tends to eliminate the large endolysosomal vacuoles generated by vacuolin-1 or apilimod and reduces the colocalization with viral particles contained within. time series with images taken every 3 s for 3 min in a single optical plane with the appropriate fluorescent channels (52) were acquired from nonfixed samples imaged at the end of the experimental period. for experiments containing npc1-halo, the halo-tagged cells were labeled with either 250 nm jf549 or jf647 dye in media for 30 min at 37°c. following labeling, cells were washed three times with media. the microscope was operated using the slidebook 6.4 software package (3i), and images were displayed also using this software. statistical tests. to compare the means from cells with different treatments, one-way anova and post hoc tukey test analysis were used to take into account unequal sample sizes as indicated in the figs. 1, 2, and 6 figure legends. data availability. the vsv virus chimeras are available from the corresponding author s.p.w. upon request. all study data are included in the article and si appendix and movies s1-s3. endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c1 sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus virus entry. lassa virus entry requires a trigger-induced receptor switch old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport coincidence detection in phosphoinositide signaling pikfyve, a mammalian ortholog of yeast fab1p lipid kinase, synthesizes 5-phosphoinositides. effect of insulin cloning, characterization, and expression of a novel zn2+-binding fyve finger-containing phosphoinositide kinase in insulinsensitive cells mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase pikfyve the mammalian phosphatidylinositol 3-phosphate 5-kinase (pikfyve) regulates endosome-to-tgn retrograde transport core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. novel sac phosphatase joins the arpikfyve-pikfyve complex functional dissection of lipid and protein kinase signals of pikfyve reveals the role of ptdins 3,5-p2 production for endomembrane integrity a selective pikfyve inhibitor blocks ptdins(3,5)p(2) production and disrupts endomembrane transport and retroviral budding vacuolin-1 inhibits autophagy by impairing lysosomal maturation via pikfyve inhibition the small chemical vacuolin-1 inhibits ca 2+ -dependent lysosomal exocytosis but not cell resealing pikfyve, a class iii pi kinase, is the target of the small molecular il-12/il-23 inhibitor apilimod and a player in toll-like receptor signaling a family of pikfyve inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis active pikfyve associates with and promotes the membrane attachment of the late endosome-to-trans-golgi network transport factor rab9 effector p40 identification of novel vacuolin-1 analogues as autophagy inhibitors by virtual drug screening and chemical synthesis vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating rab5a randomized, double-blind, placebo-controlled trial of the oral interleukin-12/23 inhibitor apilimod mesylate for treatment of active crohn's disease apilimod inhibits the production of il-12 and il-23 and reduces dendritic cell infiltration in psoriasis the phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry direct visualization of ebola virus fusion triggering in the endocytic pathway a phase 1/2a trial of sta 5326, an oral interleukin-12/23 inhibitor, in patients with active moderate to severe crohn's disease brief report: a phase iia, randomized, double-blind, placebocontrolled trial of apilimod mesylate, an interleukin-12/interleukin-23 inhibitor, in patients with rheumatoid arthritis arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses identification of combinations of approved drugs with synergistic activity against ebola virus in cell cultures characterization of vps34-in1, a selective inhibitor of vps34, reveals that the phosphatidylinositol 3-phosphate-binding sgk3 protein kinase is a downstream target of class iii phosphoinositide 3-kinase evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry identification of apilimod as a first-in-class pikfyve kinase inhibitor for treatment of b-cell non-hodgkin lymphoma the vp1 subunit of jc polyomavirus recapitulates early events in viral trafficking and is a novel tool to study polyomavirus entry the phosphoinositide kinase pikfyve promotes cathepsin-s-mediated major histocompatibility complex class ii antigen presentation small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection endosome-to-cytosol transport of viral nucleocapsids protease inhibitors targeting coronavirus and filovirus entry tracking the fate of genetically distinct vesicular stomatitis virus matrix proteins highlights the role for late domains in assembly uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin a forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry efficient recovery of infectious vesicular stomatitis virus entirely from cdna clones genome engineering using the crispr-cas9 system identification and characterization of a novel broad spectrum virus entry inhibitor a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov the first five seconds in the life of a clathrin-coated pit acknowledgments. we thank walter j. atwood for providing the parental svg-a cells; eric marino, justin h. houser, and tegy john vadakkan for maintaining the spinning disc confocal microscope (t.k. laboratory); marina cella and erica lantelme from the flow cytometry facility, department of pathology and immunology at wusm for help with flow cytometry; stephen c. harrison for helpful discussions and editorial assistance; and alex j. b. kreutzberger for editorial help. this research was supported by nih funding (grant ai109740) to s.p.j.w. and t.k., by nih maximizing investigators' research award (mira) gm130386 to t.k., by nih funding (ai059371) and unrestricted funds from wusm to s.p.j.w., and by nih contract 75n93019c00062 and nih grant r01 ai127828 to m.s.d. key: cord-318686-we6pveus authors: ehlen, lukas; tödtmann, jan; specht, sabine; kallies, rené; papies, jan; müller, marcel a.; junglen, sandra; drosten, christian; eckerle, isabella title: epithelial cell lines of the cotton rat (sigmodon hispidus) are highly susceptible in vitro models to zoonotic bunya-, rhabdo-, and flaviviruses date: 2016-05-04 journal: virol j doi: 10.1186/s12985-016-0531-5 sha: doc_id: 318686 cord_uid: we6pveus background: small mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. however, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. the cotton rat (sigmodon hispidus) is a new world rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens. methods: a method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. susceptibility to members of zoonotic bunya-, rhabdo-, and flaviviridae, in particular rift valley fever virus (rvfv), vesicular stomatitis virus (vsv), west nile virus (wnv), and tick-borne encephalitis virus (tbev) was tested. furthermore, novel arthropod-derived viruses belonging to the families bunya-, rhabdo-, and mesoniviridae were tested. results: we successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. cells were shown to be interferon-competent. viral infection assays showed high-titre viral replication of rvfv, vsv, wnv, and tbev, as well as production of infectious virus particles. no viral replication was observed for novel arthropod-derived members of the bunya-, rhabdo-, and mesoniviridae families in these cell lines. conclusion: in the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. these cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species. infectious diseases are a major threat to human health and remain among the leading causes of death and disability worldwide [1] . in the last decade, a variety of viruses such as ebola virus, hendra virus, nipah virus, west nile virus (wnv), and severe acute respiratory syndrome (sars)and middle east respiratory syndrome (mers)-coronaviruses have emerged or re-emerged, all of which are of zoonotic origin [2] [3] [4] [5] . there have been a large number of novel, potentially zoonotic viruses that have been shown to be associated with small mammals, especially those of the orders chiroptera and rodentia, [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . however, the isolation and propagation of these novel viruses has been unsuccessful in most instances, which limits further evaluation of their zoonotic risk. upon characterizing these novel viruses, it has become clear that most available animal models such as the domestic mouse or rat are of limited use, as they do not reflect the evolutionary conserved pathogen-host interaction that is a key trait of many reservoir-restricted viruses. in light of the large species range in which novel and potentially zoonotic viruses have been discovered, there remains a need for suitable in vitro models to understand virus-host interactions, interspecies spillover, and general viral pathogenicity [15] . additionally, many of the natural reservoir hosts are protected or cannot be held in captivity, which limits in vivo studies in relevant hosts. therefore, species-specific cell culture models may serve as acceptable surrogates [16] [17] [18] [19] . the cotton rat (sigmodon hispidus) is a unique example of a rodent species that is a well-established animal model to study viral pathogenesis and is also associated with a large range of zoonotic viruses in the wild [20] [21] [22] . experimental studies in cotton rats have been performed for a large variety of human viruses, including important respiratory pathogens such as influenza or parainfluenza viruses, respiratory syncytial virus, and human metapneumovirus [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . furthermore, in the wild, cotton rats are associated with a variety of known or potential zoonotic viruses, such as classical rodent-borne viruses from the genera hantavirus and arenavirus, as well as members of the family flaviviridae, such as wnv and st. louis encephalitis virus (slev) [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] . to evaluate whether the broad viral susceptibility seen in both animalmodel and wild cotton rats was also reflected in in vitro cell culture models, we generated continuous cell lines from the respiratory and renal tracts of a cotton rat, and assessed their use for virus replication studies of known and potentially novel zoonotic viruses. tissues from a laboratory-bred 3-month-old male cotton rat (s. hispidus) were kindly provided by the institute for medical microbiology, immunology and parasitology (immip), university of bonn medical centre, bonn, germany. ethical clearance was obtained from the respective authorities (no. az 84-02.05.20.13.114). the trachea and both kidneys of the euthanized cotton rat where removed in toto. all subsequent steps were then performed under sterile conditions using a laminar flow hood. briefly, organ specimens were cleaned from surrounding tissue and then either sliced or roughly chopped with a sterile blade. tissue slices were placed in 6-well cell culture plates at 37°c in primary cell media. for tracheal cells, airway epithelial cell growth medium was used containing the following supplements: 0.004 ml/ml bovine pituitary extract, 10 ng/ml recombinant human epidermal growth factor, 5 μg/ml recombinant human insulin, 0.5 μg/ml hydrocortisone, 0.5 μg/ml epinephrine, 6.7 ng/ml triiodo-l-thyronine, 10 μg/ml human holotransferrin, and 0.1 ng/ml retinoic acid (promocell, heidelberg, germany). for kidney cells, renal epithelial cell growth medium was used containing the following supplements: 0.05 ml/ml foetal calf serum (fcs), 10 ng/ ml recombinant human epidermal growth factor, 5 μg/ml recombinant human insulin, 36 ng/ml hydrocortisone, 0.5 μg/ml epinephrine, 4 pg/ml triiodo-l-thyronine, and 5 μg/ml human holo-transferrin (promocell). both media were supplemented with 1 % penicillin/streptomycin (life technologies gmbh, darmstadt, germany), 0.5 % of ofloxacin (tarivid, sanofi-aventis, frankfurt, germany) and 1 % amphotericin b (paa, pasching, austria) to avoid bacterial and fungal contamination during primary cell isolation and growth. after the outgrowth of primary cells from organ specimens, the medium was changed every 2 days, and cell outgrowth was regularly observed. when nearly confluent, cells were immortalized by lentiviral transduction of the large t antigen of sv40 as described previously [17, 19, 53] . after immortalization, cells were passaged and stock-frozen until further use. all cell cultures were genotyped by polymerase chain reaction (pcr) amplification and sequencing of the mitochondrial cytochrome c oxidase subunit i and cytochrome b oxidase subunit i genes [54, 55] . to obtain single cell clones, cells were subcloned by end-pointlimiting dilution and adapted to dulbecco's modified eagle's medium (dmem) (paa, cölbe, germany) with 4.5 g/l glucose (paa), supplemented with 10 % fcs (paa), 2 mm l-glutamine, 1 mm sodium pyruvate (paa), nonessential amino acids (neaa), 1 % penicillin/streptomycin (100x concentrate contains 10,000 units/ml penicillin and 10 mg/ml streptomycin) (life technologies), and 1 % amphotericin b as described previously [17, 19] . cells were seeded on glass slides, and were washed the next day with pbs and fixed with acetone-methanol (1:1) for 5 min. then, the acetone-methanol was removed and cells were washed again with pbs. each slide was subsequently incubated overnight at 4°c with 250 μl primary mouse monoclonal antibodies against pan-cytokeratin (abcam ab7753, cambridge, uk) and rabbit polyclonal antibodies against zonula occludens-1 (zo-1 mid) (invitrogen 40-2200, carlsbad, ca, usa) diluted 1:500 in pbs. cells were washed and then incubated for 30 min at 25°c with 125 μl cyanine 3 (cy3)-labelled donkey-antimouse and cy2-labelled donkey-anti-rabbit secondary antibodies (dianova, hamburg, germany) diluted 1:500 in pbs. cells were washed and then nuclei were counterstained with dapi diluted at 1:1000 in pbs for 1 min. all images were obtained with a 207 motic axiovision microscope (zeiss, jena, germany). immortalized s. hispidus cells were seeded in 24-well plates at a density of 4 × 10 5 cells/ml and grown in dmem containing 5 % fcs and supplements as described above. the following day, cells were infected with vesicular stomatitis virus (vsv) strain indiana or rift valley fever virus (rvfv) clone 13 at multiplicity of infections (mois) of 0.1 and 0.001 for both viruses. cells were infected with wnv strain new york or tick-borne encephalitis virus (tbev) strain k23 with mois of 0.01 and 0.001. infectious units of the viral stocks and in the supernatant at the end of each experiment were determined by plaque-assays with avicel overlays for rvfv and vsv as described previously [56] , and with agarose overlays for wnv and tbev as described previously [57] . for virus infection experiments, the medium was removed and cells were inoculated with virus diluted in optipro serum-free medium (life technologies) for 1 h at 37°c. then, cells were washed twice with pbs. growth medium was added and supernatants were harvested 0, 12 and 24 h after infection (hpi) for vsv; 0, 24 and 48 hpi for rvfv and 0, 6, 24 and 48 hpi after infection for wnv and tbev. all virus infection experiments were performed in three individual replicates. viral rna was extracted from cell culture supernatants with the nucleospin rna virus kit according to the manufacturer's instructions (machery-nagel, düren, germany). pcr was performed using the superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen). cycling conditions for vsv and rvfv quantitative reverse-transcription (qrt)-pcr were as follows: reverse transcription for 15 min at 55°c, initial denaturation for 2 min at 95°c, and 45 cycles of denaturation for 15 s at 95°c and primer annealing/elongation for 30 s at 58°c. cycling conditions for wnv qrt-pcr were as follows: reverse transcription for 30 min at 45°c, initial denaturation for 5 min at 95°c, and 45 cycles of denaturation for 5 s at 95°c and primer annealing/ elongation for 35 s at 57°c. qrt-pcr was carried out using the lightcycler 480 real-time pcr system (roche, basel, switzerland). primers and probes are available upon request. to test the susceptibility of the s. hispidus cell lines to a variety of novel arthropod-derived viruses, cells were seeded in 24-well plates at a density of 4 × 10 5 cells/ml. the following day, cells were infected with a titrated c6/ 36 cells-generated virus stock of ferak [58] , moussa [59] , or cavally [60] virus at an moi of 1.0. after infection, cells were observed daily for the presence of cytopathic effects (cpe). supernatants from all infected cells were passaged onto fresh cells every 7 days for a total of three passages. viral rna was extracted from cell culture supernatants, and the presence of specific viral rna was evaluated by qrt-pcr as described above. to assess the interferon (ifn) competence of the cells, cells were seeded in 24-well plates at a density of 4 × 10 5 cells/ml and grown in dmem containing 10 % fcs and supplements as described above. the following day, cells were either transfected in triplicates with 1 μl of total rna from vsv-infected cells (vsv-rna) using the xtreme gene sirna transfection reagent (roche, basel, switzerland) to stimulate the ifn response of the cells [61] or cells were left untreated as control. eight hours after transfection, all cells were infected with the ifnsensitive rvfv clone 13 carrying a renilla luciferase [62] . 16 h after infection, cells were treated with lysis buffer and renilla luciferase activity was measured in a microplate reader. in order to assess the role of cotton rats as an experimental animal model for viral diseases and as a reservoir of zoonotic viruses in the wild, a review of the literature was performed. all studies that described cotton rats as experimental animal models for viral research, and all studies that described an association between viruses (via direct detection by pcr, or viral isolation in cell culture and antibody findings) and wild cotton rats were included ( table 1) . outgrowth of primary airway and kidney epithelial cells from cotton rat tissue samples was observed 3-5 days after the initiation of the cell culture. outgrowing cells displayed a homogeneous, cobblestone-like morphology typical of epithelial cells in both the airway and renal epithelial cell cultures (fig. 1) . successful immortalization was achieved by lentiviral transduction of the large t antigen of sv40 when the first patches of primary cells were visible in the cell culture dishes. both airway epithelial (subsequently termed shispaec.b) and renal epithelial (subsequently termed shisprec.b) cell lines showed rapid increases in cell growth 1-2 weeks after immortalization. to generate a homogeneous cell line, subclones were obtained and further characterized. by endpoint-limiting dilution, single-cell clones were selected and two subclones were used for further experiments, which were subsequently termed shispaec.b-2 and shisprec.b-6. both of these cell lines displayed epithelial cell morphology. the immortalized cell lines and the subclones generated in this study showed expression of pan-cytokeratin and zonula occludens 1 protein, confirming that the cells were of epithelial origin. cytochrome b pcr amplification and sequencing of the product confirmed the host species (data not shown). shispaec.b-2 and shisprec.b-6 were tested for their ability to respond to external stimulation of the interferon system. in order to stimulate the ifn response cells were transfected with total rna from vsv-infected cells which was shown to trigger the rig-i and mda5dependent ifn signalling cascade [61] . in comparison to venezuelan equine encephalitis virus [40] [41] [42] eastern equine encephalitis virus [36] untreated cells (fig. 2, light column) , vsv-rna transfected cells (fig. 2 , dark columns) showed a 10-fold (renal cells) to 500-fold (airway cells) reduced replication of a highly ifn-sensitive rvfv-renilla reporter virus. the pronounced decrease of rvfv-renilla replication reflects the efficient induction of an antiviral state in both cell cultures. overall, these data show that both subclones harbour an intact ifn response to external stimulation with airway cells showing a higher stimulation than renal epithelial cells. shispaec.b-2 and shisprec.b-6 were infected with vsv and rvfv with two different mois, and the supernatants were harvested at different time points (fig. 3) . vero e6 cells served as controls and were treated in parallel. both cell lines exhibited a cpe and cell death after vsv and rvfv infection (data not shown). a 5.0 log increase in vsv viral rna genome equivalent (ge) copies was seen after infection with an moi of 0.1 for the airway epithelial cells, and a 4.1 log increase in ge copies was observed for the renal epithelial cells (fig. 3a) . vero e6 cells showed an increase in ge copies of almost 6 log with the same experimental set-up. upon infection with an moi of 0.001, the maximum increases in log ge copies were approximately one log lower than those with an moi of 0.1, with the highest ge copy numbers reached in vero e6 cells (5.3 log increase), followed by the airway epithelial cells (4.1 log increase), and the renal epithelial cells (3.5 log increase). production of infectious vsv particles was assessed 24 h after infection by titration of supernatants on vero e6 cells, resulting in more than 8 log pfu/ml in all three cell lines after infection with a moi of 0.1 and 0.001 (fig. 3b ). upon infection with rvfv at an moi of 0.1, a maximum increase of 2.2 log in viral rna ge copies was observed in the airway epithelial cells, and a 2.9 log increase in ge copies was seen for the renal epithelial cells. vero e6 cells showed an increase of 2.7 log ge copies. infections with a lower moi of 0.001 showed comparable growth kinetics with slightly lower maximum increases in viral rna (fig. 3c) . production of infectious rvfv particles was assessed 48 h after infection by titration of supernatants on vero e6 cells. the highest number of plaque-forming units was seen in shis-prec.b-6 with 6.7 log pfu/ml, followed by vero e6 and shispaec.b-2 with 6.0 log and 4.5 pfu/ml after infection with an moi of 0.1. comparable results were observed after infection with a lower moi resulting in 5.7; 5.1 and 3.7 log pfu/ml, respectively, for shisprec.b-6, vero e6 and shispaec.b-2 (fig. 3d) . to assess s. hispidus cell susceptibility to viruses from the flaviviridae family, infection experiments with wnv strain new york and tbev were performed with two different mois, and the supernatants were collected at different time points. vero e6 cells served as controls and were treated in parallel. upon infection with tbev, both s. hispidus cell lines and vero e6 cells showed a cpe and rapid cell death within 48 h (data not shown). the maximum increase in viral rna ge copies was 7.2 log for the airway epithelial cells, 7.6 log for the renal epithelial cells, and 8.4 log for vero e6 cells after infection with a moi of 0.01. comparable growth kinetics were seen after infection with a moi of 0.001. production of infectious tbev particles was assessed 48 h after infection by titration of supernatants on bhk-j cells. tbev infectious particles were produced by all cell lines in comparable amounts of approximately 6 log pfu/ml after infection with a moi of 0.01 and 0.001 (fig. 4b) . for wnv, no increase in viral rna was seen for the airway epithelial cells at either moi. for the renal epithelial cells, a maximum increase of viral rna ge copies of 2.2 log was observed, and a 4.0 log increase in ge copies was seen for vero e6 cells. comparable growth curves were seen for the lower moi of 0.001 production of infectious wnv particles was assessed 48 h after infection by titration of supernatants on bhk-j cells. the highest number of plaque-forming units was seen in veroe6 cells with approximately 6 log pfu/ml, followed by lower titers in shis-prec.b-6 with 4.0 and 4.6 pfu/ml after infection with an moi of 0.01 and 0.001, respectively. no production of infectious particles was seen in shispaec.b-2 cells (fig. 4d) . to assess the susceptibility of s. hispidus cell lines to members of the rhabdo-, bunya-, and mesoniviridae families, further infection experiments were performed with recent novel virus isolates from insects [58] [59] [60] . both airway and renal epithelial cells were inoculated with isolates of moussa, ferak, and cavally viruses with a moi of 1. no cpe was seen with daily observation. supernatants were tested by viral specific qrt-pcr at the end of each passage, which did not reveal an increase in viral rna, thus arguing against replication of these viruses in the cell lines generated in this study (fig. 5 ). in the work presented herein, we generated epithelial cell lines from the respiratory and renal tracts of a cotton rat due to its susceptibility to a broad range of human viruses, as well as the association of multiple important and emerging zoonotic viruses with this species. s. hispidus is a rodent species with a long-standing history as an experimental animal model for virus research. although the first animal experiments on cotton rats date back to the 1940s, only two cell lines from cotton rats are available to date. however, in contrast to experimental animals, cell lines are a less laborious model system, less expensive, and can be used in large-scale viral experiments such as in virus isolation trials without the ethical considerations that are involved in animal experiments. from the cotton rat, an osteoblastic cell line was previously derived from an osteogenic sarcoma (ccrt), of which two lymphoid cell lines (cr-t 1 and cr-t 2) were also derived [22] . however, these cell lines are used for the induction of tumours and as hybridoma cells to produce antibodies. no evaluation of these cells for their use in virus research has been performed despite a large range of viruses that have been investigated in s. hispidus animal experiments. moreover, these cell lines are tumour cells that may not adequately resemble cells in vivo to study virus-host interactions, and they are not derived from target cells that are relevant to the natural course of a viral infection, such as epithelial cells. the value of species-specific cell lines has been shown particularity in the field of bat-borne viruses, where the use of bat cell lines has contributed significantly to studies of novel viruses, virus evolution, and virus adaptation during cell culture as well as replicative capacity and expression of host receptors [16, 53, 56, [63] [64] [65] (for a review see [18] ). cell lines derived from potential reservoir and intermediate hosts can serve as a valuable surrogate to study the replicative capacity of emerging zoonotic viruses, as has been demonstrated for the recently emerged viruses mers-cov and ebola virus by work from our group [16, 56, 63, 66, 67] . in the current study, we evaluated the replicative capacity of viruses belonging to the four families bunyaviridae, rhabdoviridae, flaviviridae, and mesoniviridae in s. hispidus epithelial cell lines. several members of the bunyaviridae family were already shown to infect cotton rats, including black creek canal virus (bccv), which belongs to the genus hantavirus. this virus was isolated from the lungs and spleens of cotton rats, and it was further shown by serologic analysis that s. hispidus was the primary rodent reservoir of bccv [34, 35] . other hantaviruses associated with s. hispidus are bayou virus and muleshoe virus [37, 39] . additionally, from the genus orthobunyavirus, an isolate termed zegla virus was obtained from s. hispidus [68] . here we showed that s. hispidus epithelial cells are highly susceptible to rvfv, a bunyavirus belonging to the genus phlebovirus, with comparable growth kinetics to interferon-deficient vero e6 cells. furthermore, we tested the susceptibility of s. hispidus cells to a recently isolated bunyavirus termed ferak virus that belongs to the sister taxon of the genus orthobunyavirus. interestingly, no growth of this virus was seen in the s. hispidus cell lines, suggesting an insectspecific replication cycle for this virus [58] . the further use of these cell lines for rodent-associated bunyaviruses such as hantaviruses should be evaluated in light of the promising findings for rvfv demonstrated herein. for the rhabdoviridae family, there have been serological findings in cotton rats that suggest a role for this species in the natural cycle of these viruses. specifically, it was shown that neutralizing antibodies to both indiana and new jersey serotypes were found in s. hispidus in a vsv enzootic area in costa rica. antibodies against either one or both serotypes were only found in s. hispidus, and not in exposed mus musculus [49] . our in vitro results showed that vsv replicates readily in s. hispidus cell lines with high replication titres of up to almost 11 log ge copies, which is approximately only one log lower than that of the replication titres seen in vero e6 cells. these findings suggest that the s. hispidus cell culture models could serve as suitable in vitro models for further studies on vsv. to further assess the replication capacity of other rhabdoviruses in s. hispidus cells, the insect-derived moussa virus was used [59] . moussa virus was isolated from mosquitoes that also feed on mammals, but thus far, viral replication in human, hamster, or porcine cells has not been successful [59] . however, in line with the findings of a study by quan et al., no replication of moussa virus was seen in our experiments with s. hispidus cells. additionally, another insect-derived isolate of a novel virus family termed mesoniviridae was tested on our s. hispidus cell lines. here, no replication of cavally virus on the newly generated cells was seen. a strong association has been reported between the cotton rat and several zoonotic flaviviruses, including wnv, slev, san perlita virus, and cowbone ridge virus [51, 52, 69, 70] . furthermore, cotton rats have been discussed as potential reservoir hosts in the wild for arboviruses, by which infected viremic cotton rats serve as a reservoir for arthropods that feed on them. in our cell culture experiments with tbev and wnv, we saw replication and production of infectious virus particles in both s. hispidus cell lines for tbev and in the kidney epithelial cells for wnv. moreover, both replication titres were only one log lower than that for vero e6 cells, indicating a high susceptibility of these cell lines to flaviviruses. additionally, we have used s. hispidus cell lines for the evaluation of a novel sylvatic isolate of slev in an earlier study [71] . here, it was shown that the endemic strain of slev, termed msi-7, replicated in s. hispidus kidney cells. in contrast, the novel sylvatic slev isolate, termed palenque strain, did not show any replication. as s. hispidus has been described as a natural reservoir host for slev, these findings suggest that the sylvatic isolate has not yet adapted to hosts that live outside the primary rain forest, whereas the endemic strain has [71] . in line with the findings obtained for slev, our results showed that wnv only replicated in kidney cells but not in airway epithelial cells, suggesting that kidney cells are more susceptible to this virus than airway cells. taken together, the multiple in vitro findings presented herein for flaviviruses provide evidence that cotton rats may be reservoirs for multiple members of flaviviridae in the wild. therefore, s. hispidus cell lines, especially s. hispidus kidney epithelial cells, may provide a useful model for in vitro virus-host interaction studies. newly generated epithelial cell lines from s. hispidus are able to support the replication of virus species from important zoonotic virus families, and may therefore serve as valuable tools for studies focusing on the isolation of novel viruses and virus-host interactions. emerging infectious diseases: a 10-year perspective from the national institute of allergy and infectious diseases the challenge of emerging and reemerging infectious diseases global trends in emerging infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia 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poliomyelitis virus in cotton rats and white mice hiv type-1 infection of the cotton rat (sigmodon fulviventer and s. hispidus) we thank marco marklewitz, pascal trippner and anne kopp for help with novel arthropod-derived viruses; beate kümmerer and janett wieseler for viral isolates of wnv and tbev; and bettina dubben for organ samples. we thank friedemann weber (university of gießen) for providing rvfv clone 13 and rvfv-renilla reporter virus and alexander pfeifer, (university of bonn) for providing large t antigen lentiviruses. the work was funded by the german research platform for zoonoses and the federal ministry of education and research (grant no. 01ki1308 to ie). the authors declare that they have no competing interests.authors' contributions ie, le, mam, sj, and cd designed the experiments; le, jt, jp and rk performed experiments and analyses; le and ie wrote the manuscript; and all authors contributed to, read, and approved the final version of the manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-267712-mhx8e5y0 authors: fang, xinkui; zhang, shikuan; sun, xiaodong; li, jinjin; sun, tao title: evaluation of attenuated vsvs with mutated m or/and g proteins as vaccine vectors date: 2012-02-08 journal: vaccine doi: 10.1016/j.vaccine.2011.12.085 sha: doc_id: 267712 cord_uid: mhx8e5y0 vesicular stomatitis virus (vsv) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. in experimental animals (primates, rats, and mice), vsv has been shown to lead to neurotoxicities, such as hind limb paralysis. the virus matrix protein (m) and glycoprotein (g) are both major pathogenic determinants of wild-type vsv and have been the major targets for the production of attenuated strains. existing strategies for attenuation included: (1) deletion or m51r substitution in the m protein (vsvδm51 or vsvm51r, respectively); (2) truncation of the c-terminus of the g protein (gδ28). despite these mutations, recombinant vsv with mutated m protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant vsv with truncated g protein at high dose. thus, a novel recombinant vsv (vsvδm51-gδ28) as well as other attenuated vsvs (vsvδm51, vsv-gδ28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. in vitro studies indicated that truncated g protein (gδ28) could play a more important role than deletion of m51 (δm51) for attenuation of recombinant vsv. vsvδm51-gδ28 was determined to be the most attenuated virus with low pathogenicity in mice, with vsv-gδ28 also showing relatively reduced pathogenicity. further, neutralizing antibodies stimulated by vsv-gδ28 proved to be significantly higher than in mice treated with vsvδm51-gδ28. in conclusion, among different attenuated vsvs with mutated m and/or g proteins, recombinant vsv with only truncated g protein (vsv-gδ28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. these properties make vsv-gδ28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease. vesicular stomatitis virus (vsv) is a single-strained negativesense rna virus that has been widely used as a vector for vaccine development [1, 2] . recombinant vsv can accommodate large and multiple foreign genes in its genome that are expressed at high levels [3] and confer advantages over other rna viral vectors. due to its potent capabilities in triggering cellular, humoral, and mucosal immunities in animals, even after a single administration, recombinant vsv has been studied as a vaccine vector not only for preventing vesicular stomatitis disease in livestock [4] , but a number of human pathogens including: influenza virus, ebola virus, marburg virus, human immunodeficiency (hiv) virus, severe acute respiratory syndrome (sars) virus, and hepatitis c virus [5] [6] [7] [8] [9] . however, vsv is a notoriously infectious agent that not only produces acute disease, such as vesicular lesions in cattle, swine and horses, but neurotoxicity in experimental animals, including primates and mice [10] [11] [12] . therefore, modifications are needed to improve the safety of vsv before it can be applied clinically as a replication competent vector. vsv genomic rna is transcribed into five capped and polyadenylated mrnas by the viral rna-dependent rna polymerase. the mrnas encode five structural proteins: nucleocapsid protein (n); phosphoprotein (p), which is a cofactor of the rna-dependent rna polymerase (l); matrix protein (m); and attachment glycoprotein (g) [12] . m and g proteins are both primary pathogenic determinants of vsv [13, 14] . the m protein is a multi-functional protein involved in virus assembly, budding and pathogenesis [15, 16] , and capable of inhibiting the transport of host mrnas out of the nucleus significantly inhibiting type i interferon (ifn) signaling [17] . the g protein is responsible for viral binding to the host receptor and entry of vsv into host cells and its cytoplasmic domain is 0264-410x/$ -see front matter © 2011 elsevier ltd. all rights reserved. doi:10.1016/j.vaccine.2011.12.085 considered to play an important role in viral budding and packaging [18] [19] [20] . to date, several strategies have focused on the m or g proteins for the generation of attenuated vsv. it was reported that deletion of methionine residue 51 (vsv m51) or m51r substitution (vsvm51r) within the m protein can lead to attenuation due to the potent induction of type i ifn [17, 21] . another attenuation strategy is dependent upon truncation within the c-terminal region of the g protein (vsv-g 28) that significantly impacts viral budding and packaging efficiency [22] . in vivo, however, vsv m protein mutant proved to be only moderately attenuated in experimental infections [16, 21] , whereas there is currently no information available if recombinant vsv with truncated g protein is safe or not when animals challenged with high dose of the mutant virus. the current study developed a novel recombinant vsv (vsv m51-g 28) with mutations in both m and g proteins, including deletion of methionine 51 in the m protein and a truncation of 28 amino acids in c-terminal region of the g protein. it was hypothesized that vsv m51-g 28 could combine the advantages observed for the m51 and g 28 mutations to produce a safer and more effective vaccine vector. furthermore, for the first time, different attenuated vsvs with mutated m and/or g proteins were comprehensively compared in vitro and in vivo. based on pathogenicity and capabilities to stimulate potent immune responses, we aimed to identify a suitable recombinant vsv vaccine vector and vaccine candidate for preventing vesicular stomatitis disease. the recombinant vsvs rescued in our study were based on the infectious clone, vsv indiana [1] . the plasmid pvsv m51 was kindly provided by prof. john bell (univ. of ottawa, canada). the pvsv xn2 and helper plasmids, pbs-n, p, l, were provided by prof. glen barber's lab (univ. of miami, usa). the pvsv-g 28 and pvsv m51-g 28 were constructed based on pvsv xn2 and pvsv m51, respectively. in the constructs, the wild-type vsv g protein cytoplasmic tail (29 aa) was truncated with only one arginine residue remaining in the tail (g 28) (fig. 1 ). the forward primer used for polymerase chain reaction (pcr) amplification of g 28 was: 5 -ccggagcgctatgaagtgccttttgtactta-3 (underlined indicates the mlui restriction site), and the reverse primer was: 5 -cc-ggctcgagcgtgatatctgttagtttttttcatacctagcaggatttg-agtcattatcgggagaaccaagaatag-3 (underlined indicates the xhoi restriction site and bold residues indicate the two tandem repeat stop codons). to construct pvsv-g 28 or pvsv m51-g 28, vsv g gene of pvsv xn2 or pvsv m51 was substituted with g 28 by restriction digestion with mlui and xhoi. all the start/stop signals for viral gene transcription were preserved. a schematic representation of pvsv m51-g 28 is shown in fig. 1 . recovery of recombinant vsvs from the infectious clones was performed as previously described [3] . briefly, co-transfection of vsv constructs (pvsv m51, pvsv-g 28, pvsv m51-g 28) with helper plasmids, pbs-n, p, and l, was performed into bhk21 cells infected with a recombinant vaccinia virus (vtf7-3) expressing t7 rna polymerase. at 48 h post-transfection, culture supernatants were collected and filtered through a 0.2 m filter into fresh bhk21 cells. cells were checked daily. if typical cytopathic effect (cpe) was observed 2-3 days after vsv infection, supernatants were collected and viruses were plaque-purified in vero cells. individual plaques were isolated and seed stocks were amplified in bhk21 cells. recombinant viruses were concentrated by ultracentrifugation at 30,000 rpm/min for 2 h and frozen at −70 • c. viral titer was determined by plaque assay performed in vero cells. vsv xn2 with wild-type g (wt g) and m proteins was prepared from the stock kept in our laboratory. to identify rescued recombinant vsvs, western blot was used to identify typical bands for l, g, n/p, and m proteins using specific convalescent sera from vsv xn2 -infected mice. in short, confluent bhk21 cells were infected with vsv m51, vsv g 28 or vsv m51-g 28 at a multiplicity of infection (moi) of 1. vsv xn2 was used as a control. at 24 h post-infection (p.i.), cells were lysed in buffer containing 5% ␤-mercaptoethanol, 0.01% np-40, and 2% sodium dodecyl sulfate (sds). proteins were separated by 12% sds polyacrylamide gel electrophoresis (page) and transferred to polyvinylidene fluoride (pvdf) membrane (thermo scientific, rockford, il, usa) in a mini trans-blot electrophoretic transfer cell (bio-rad, hercules, ca, usa). sera from a vsv xn2 infected mouse was used as the primary antibody at a dilution of 1:2000 with horseradish peroxidase-conjugated goat anti-mouse igg as the secondary antibody at a dilution of 1:5000 (santa cruz, ca, usa). target bands were observed using west pico chemiluminescent substrate (thermo scientific) and exposed to kodak biomax mr film (kodak, rochester, ny, usa). attenuation of virus could be characterized by analyzing plaque formation sizes in cells [16] . the a549 cell line was used as a type i ifn signaling competent cell line [23] , whereas vero cells were used as an incompetent cell line [24, 25] . plaque formation experiments were set up in these cells to identify the role by type i ifns in viral attenuation. briefly, 90% confluent a549 or vero cells in 12-well plates were infected with optimally diluted vsv m51-g 28,vsv m51, vsv g 28, or vsv xn2 and then covered with low melting temperature agar (invitrogen, carlsbad, ca, usa) after rinsing with phosphate buffered saline (pbs). at 24 h p.i., 1% crystal violet was used to stain vero cells, whereas a549 cells were stained 48 h p.i. plaques were first scanned with gel imager (tanon-1600r, tanon, shanghai, china) under bright light and then individual plaques were viewed and photographed under 4× magnification using a microscope (nikon, tokyo, japan) equipped with a digital camera. the mean plaque size was determined by measuring the area of each plaque in each group using nikon nis-elements br software (nikon). the human prostate cancer cell line, pc3, was used as another cell line with competent type i ifn signaling [17] . at 70% confluency, pc3 cells in 24-well plates were infected with viruses at a low moi of 0.01. after 1 h of absorption, the inoculum was removed and cells were washed three times with pbs, fresh dulbecco's modified eagle's medium (dmem supplemented with 2% fetal bovine serum) was added, and the infected cells were incubated at 37 • c. aliquots of cell culture supernatants were removed at 24, 48, and 72 h p.i. viral titers in supernatants were detected by plaque assays in vero cells. the harvests at 24 h p.i. were also assayed for ifn-␤ level using the human interferon-␤ elisa kit (r&d systems, minneapolis, mn, usa). specific pathogen free (spf) female balb/c mice (∼20 g body weight) were purchased from shanghai slac experimental animal company (chinese academy of sciences, china) and divided into five groups (10 per group) inoculated intranasally with vsv m51-g 28, vsv-g 28, vsv m51, vsv xn2 , or pbs under light anesthesia with ketamine-xylazine. the inoculated dose of different vsvs was 10 7 plaque-forming units (pfu) in 20 l of pbs; the highest dose that could be prepared by ultracentrifugation for vsv-g 28 or vsv m51-g 28. animal body weight losses and survival were monitored every day until 14 days p.i. all animal studies were performed in accordance with a protocol approved by the shanghai jiaotong university experimental animal center. since wild-type vsv possesses neurotropism that could lead to serious neurotoxicities in animals, recovery of vsv in brain tissues was performed to identify the associations of pathogenesis with viral replications in host. in short, spf mice were divided into five groups and were challenged intranasally with vsv xn2 , vsv m51, vsvg 28 or vsv m51-g 28 at a dose of 10 7 pfu, and the control group with pbs. in each group, two mice were sacrificed at 2 and 5 days post-inoculation and their brains were removed for viral detection. tissues were weighed in 2 ml cell frozen vials, quickly frozen in liquid nitrogen and then kept under −70 • c. for viral assay, tissues were thawed and suspended in 1 ml pbs and then completely homogenized using a dounce homogenizer. the homogenized tissues were centrifuged at 10,000 rpm for 5 min and the suspensions were serially diluted in pbs. viral titers were determined by standard plaque assays in vero cells, as described above. plaques were observed and counted after 24 h incubation. immune responses induced by vsv m51-g 28 or vsv-g 28 were evaluated in balb/c mice (∼20 g each). female spf balb/c mice were divided into three groups and immunized with vsv m51-g 28, vsv-g 28 or pbs as the control. animals in vsv m51-g 28 or vsv-g 28 treated groups were further divided into two subgroups (five per subgroup) that were housed in isolation hutches and intranasally inoculated once with viruses at doses of 10 3 pfu or 10 4 pfu in 20 l pbs. blood was collected from anesthetized mice by retro-orbital bleeds before and 21 days after vaccinations. the vaccinated mice were then challenged with vsv xn2 . blood was allowed to clot at room temperature and sera were collected after centrifugation, and stored at −80 • c for neutralization assays. sera from animals were heat inactivated at 56 • c for 30 min. those from vsv vaccinated animals were serially diluted two-fold in dmem starting at 1:5. for determination of neutralizing antibody titer, 50 l of the diluted sera were then mixed with 100 pfu vsv xn2 in 50 l dmem. the mixtures were incubated for 1 h at 37 • c before being added to 60-70% confluent bhk21 cells. cells were incubated at 37 • c for at least 2-3 days and checked for cpe. additionally, 50 l sera from pbs treated animals were directly combined with 100 pfu/50 l vsv xn2 and treated as for other sera. for titer determination, the reciprocal of the dilution giving a 100% inhibition of cpe was recorded. at 21 days after immunization, all mice were intranasally administered 10 7 pfu vsv xn2 in 20 l of pbs. animals were observed every day to calculate survival curves and body weight loss, as previously described. plaque areas, ifn-␤ concentrations, and body weight loss among different groups were compared by student's t-test, with a twotailed distribution using the statistical features in microsoft excel (microsoft, redmond, wa, usa). all viruses rescued in the current study were based on the vsv indiana infectious clone [1] . to construct safer recombinant vsv, vsv m51-g 28 was successfully generated that, for the first time, incorporated double mutations in m and g proteins. the novel virus and other attenuated vsv (vsv m51, vsv-g 28) were identified through western blotting with convalescent serum from mice infected with vsv xn2 was used as the wild-type vsv control. bands representing vsv structural proteins were identified (fig. 2) . it was proved that although length of g protein cytoplasmic domain was critical for viral budding and packaging [19] , we did find that with only one arginine residue remaining in the g protein cytoplasmic domain, vsv could still be rescued. in comparison with wild-type vsv g protein, a low molecular weight band presumed to be the g 28 was detected (fig. 2 ). viral rna of the different recombinant vsvs (vsv m51-g 28, vsv m51, and vsv-g 28) were extracted and reverse-transcription (rt)-pcr was used to amplify viral m or g genes. the pcr products were sequenced for mutations occurring in m and g genes (data not shown). two methods were used to assess attenuation of vsv m51-g 28, which included plaque formation size and multi-cycle growth curves in type i ifn signaling competent cells (a549, pc3) [17, 23] or incompetent cells (vero) [24, 25] . the results were compared to those produced by other recombinant vsvs. crystal violet stained plaque sizes were determined for vero or a549 cells infected with different vsvs (figs. 3a and 4a) . in vero cells, the average plaque sizes (n = 10) between vsvs with wt g (vsv xn2 and vsv m51) were similar (p > 0.05), but both were significantly larger than those containing the g 28 mutation (vsv-g 28 or vsv m51-g 28) (p < 0.01; fig. 3a and b) . average plaque sizes for vsv-g 28 and vsv m51-g 28 were comparable. these data indicated that, in type i ifn signaling incompetent vero cells, g 28 but not m51 was involved in vsv attenuation. in a549 cells, the average plaque areas produced by vsv xn2 were much larger than those by vsv m51, however, areas by vsv m51 was also larger than vsv-g 28 (p < 0.01; fig. 4a and b) . the smallest and turbid plaques were formed by vsv m51-g 28 in a549 cells and were difficult to detect. based on these results, both g 28 and m51 assist the attenuation of vsvs in type i ifn signaling competent a549 cell and the attenuation tendencies as follows: vsv xn2 > vsv m51 > vsv-g 28 > vsv m51-g 28. pc3 was also used in the current study as a type i ifn signaling competent cell line [17] . to quantify the relationship between amount of vsv-induced type i ifn and viral replication titers, multicycle growth curves were performed with inoculation of viruses at low moi of 0.01. as shown in fig. 5a , titers of vsv m51-g 28 reached the highest levels at 24 h p.i., but only around 5 × 10 3 pfu/ml and decline thereafter. vsv m51 also reached the ifn-␤ levels in vsv-treated pc3 cells were quantified at 24 h p.i. to identify the role by type i ifns. in different recombinant vsv-infected pc3 cells, induction of ifn-␤ was inversely correlated with viral replication titers. as shown in fig. 5b , the high levels of ifn ␤ were induced in m51 vsv-treated cells. in vsv m51 infected cells, ifn-␤ concentrations reached ∼2000 pg/ml, but the highest replication titer was ∼2 orders of magnitude lower than for vsv xn2 . although ifn-␤ production in vsv m51-g 28 treated cells was as low as ∼300 pg/ml, this was still significantly higher than vsv xn2 (p < 0.05). ifn-␤ detected in the supernatants of vsv-g 28 treated cells was below the limit of detection for the assay. therefore, viral attenuation showed the following trends: vsv xn2 > vsv m51 > vsv-g 28 > vsv m51-g 28. importantly, the fact that m51 led to attenuation of vsv through the induction of antiviral ifns was proved again in our study. based on the above data, both g 28 and m51 were shown to be involved in the attenuation of vsv, however, g 28 could play a more important role than m51. regardless, the double mutation vsv, vsv m51-g 28, showed the most significant attenuation in vitro. the current study investigated the pathogenesis of vsv m51-g 28 in balb/c mice and compared with other attenuated vsv or vsv xn2 as the wild-type virus control. inoculation of vsv through intranasal has been proven to be the most sensitive way to evaluate pathogenesis by vsv [10, 22] . a challenge dose of 10 5 -10 6 pfu wild-type vsv has been previously shown to result in significant mortality in balb/c mice [10] . therefore, in our studies, spf balb/c mice were inoculated intranasally with different recombinant vsvs at a dose of 10 7 pfu/20 l; the maximum dose that could be prepare for concentrated vsv-g 28 or vsv m51-g 28 in a 20 l volume. body weight and survival curve were used as indications of pathogenesis and monitored every day for 14 days p.i. as shown in fig. 6a , the body weight of naïve mice treated with vsv m51-g 28 or vsv-g 28 increased steadily post infection without death, similar to the pbs group. however, in the vsv xn2 treated group, mice lost significant body weight (∼30%) and neurotoxicities were evident in some animals ( fig. 6a and c) with all animals dying at around 6 days p.i. (fig. 6b) . vsv m51 showed moderate attenuation compared with vsv xn2 , but infected mice still showed significant loss of body weight with 40% mortality. wild-type vsv possesses neurotropism in many animals, including primates, rats, and mice that can lead to serious neurological deficits, such as hind limb paralysis. to identify the association of pathogenesis with viral replication, naïve mice were inoculated intranasally with different vsvs at a dose of 10 7 pfu. viruses isolated from the brains of animals were screened by plaque assay at 2 and 5 days p.i. as shown in table 1 , vsv xn2 treated mice showed viral titers up to 4 × 10 5 pfu/g at 2 days p.i and 1.6 × 10 3 pfu/g at 5 days p.i. although much lower than vsv xn2 , viral titers detected in brain tissues from vsv m51 infected mice were determined to be 3.5 × 10 2 pfu/g at 2 days p.i., but no virus was detected at 5 days p.i. in vsv m51-g 28 and vsv-g 28 treated mice, no virus could be detected in brain tissues at the two time points, which could explain why animal body weight kept steadily increased over the duration of the study in these groups. therefore, this study showed that virulence of different vsvs was closely related to replication levels in the target organ, namely the brain. vsv m51 was not as safe as those with truncated g proteins in vivo. vsv m51-g 28 and vsv-g 28 were attenuated enough as vaccine vectors. recombinant vsvs with truncated g protein (vsv m51-g 28, vsv-g 28) have been proved to be significantly attenuated both in vitro and in vivo, whereas the attenuation of the viral vector was often associated with the loss of vector immunogenicity. therefore, an ideal viral vector should retain both essential characteristics to be considered safe and effective. to evaluate protective immunity stimulated by vsv m51-g 28 or vsv-g 28 in vivo, spf mice were immunized at different doses and then challenged with a lethal dose of vsv xn2 . blood was taken before and 21 days post immunization for neutralization antibody assays. as shown in fig. 7 , the vsv m51-g 28 immunized group immunized with 10 4 pfu showed survival of all animals after challenge (fig. 7a) , however, mice still lost body weight up to ∼20%. at an immunization dose of 10 3 pfu, 40% of animals died at around 6 days post-challenge and all animals suffered serious body weight loss up to 25% and recovered very slowly (fig. 7b) . in mice immunized with vsv-g 28 at doses of 10 3 or 10 4 pfu, the body weights of all animals increased steadily after challenge with no death and significant body weight loss. all animals in pbs control group died at 6 days post-challenge (fig. 7c) . therefore, these data suggested that vsv-g 28 could stimulate more potent protective immunities than vsv m51-g 28. to further characterize the protective response, neutralization antibodies developed in the vaccinated animals were assayed. as shown in table 2 , no antibody titer could be detected in animals before vaccination and pbs treated mice. it was determined that the antibody titer generated by immunization with vsv-g 28 against the parental, vsv indiana , was significantly higher than those produced by vsv m51-g 28 immunization. in vsv-g 28 groups, antibody titers were 320-640 at a vaccination dose of 10 3 pfu and 640-1280 at 10 4 pfu. however, in the vsv m51-g 28 group with a dose of 10 3 pfu, antibody titer was only 10-20 and all mice with a titer of 10 died post-challenge (fig. 7b) indicating that the titer was unable to protect animals. moreover, even animals vaccinated at 10 4 pfu, antibody titer only reached 40-80. based on these results, it could be concluded that vsv-g 28 could stimulate a more potent protective humoral immune response than vsv m51-g 28, which might be due to the extreme attenuation of vsv m51-g 28 in vivo. as a result, vsv-g 28 may attain an ideal balance between pathogenesis and stimulating a protective immune response and with the potential as a vaccine vector and vaccine candidate for vesicular stomatitis disease. replication-competent viruses with little or no pathogenesis have been important alternatives for developing vaccines or vaccine vectors due to their capabilities in stimulating potent and comprehensive immunity in vivo. however, safety and efficacy are equally important criteria. traditionally, the isolation of spontaneously attenuated virus in host or serial passages of wild-type virus in a non-natural host were important methods to acquire attenuated viruses. with the development of molecular technology, the construction of recombinant viruses based on reverse genetics, have become common for developing novel viral vectors. vsv is a promising vector for both vaccination and oncolysis, but still has not been applied clinically. one of the main reasons lies in its potential pathogenesis in humans and animals. rearrangement of structural protein genes [26] and mutations in m or g proteins are existing strategies to generate attenuated vsv that are replication competent. our study focused on the two pathogenic proteins of vsv, m and g proteins, for the purposes of: (1) making a safer vsv; (2) table 1 recombinant vsvs recovered from brain tissues of mice (pfu/g). naïve mice were inoculated with recombinant vsvs including vsvxn2, vsv m51,vsv-g 28,vsv m51-g 28 or pbs as a control. the brains of two mice were removed at 2 or 5 days post-inoculation. homogenized brains were assayed for viral titers and the mean values were calculated. nd: not detected. viral titers in mice brain (pfu/g) vsv m51 was attenuated in vivo. on the other hand, vsv is an enveloped virus that is released via budding from host cell membranes. previous studies have noted that the length of cytoplasmic domain on vsv g, not the specific sequence, was required for efficient viral budding [19] . the real role of truncated g protein in attenuation of vsv has still not been identified, although it was reported that vsv c-terminal truncated g proteins acquired complex oligosaccharides ∼6-fold slower than for wt g protein and displayed a reduced rate of transport from the endoplasmic reticulum to the golgi apparatus, and presumably to the cell surface [18] . through in vitro assays in type i ifn signaling competent cells/incompetent cells, both m51 and g 28 were proven to be important for vsv attenuation. in vero cells, g 28 but not m51, was involved in vsv attenuation. recombinant vsv with g 28 was shown to form significantly smaller plaques than virus with wild-type g, whereas plaque sizes between vsv xn2 and vsv m51 or those between vsv-g 28 and vsv m51-g 28 were similar, no matter whether m protein was mutated or not. however, in a549 cells with effective type i ifn signaling, attenuation tendencies were demonstrated follow these tendencies: vsv xn2 > vsv m51 > vsv-g 28 > vsv m51-g 28. this tendency was also proven in pc3 cells through multi-cycle growth curve characterization. the replication titers of different vsvs were shown to be inversely correlated with the expression levels of type i ifns. vsv m51-g 28 formed the smallest plaque in a549 cells and lowest titer in pc3 cells among all vsvs tested in the current study due to the double mutations in m and g proteins. this study developed two main conclusions both in vitro and in vivo studies: (1) both g 28 and m51 mutations assisted attenuation of vsv, and (2) g 28 could play a more important role than m51 for attenuation. therefore, vsv m51 was not suitable as a vaccine vector due to its potential pathogenesis in animals and safety is the most important criteria for developing a vaccine or vaccine vector. on the other hand, vsv m51 may be suitable for virotherapy, as many studies have attempted [27, 28] , since, (1) efficacy is a priority as an antitumor drug and (2) many types of tumor cells are type i ifns signaling defective [29] . therefore, as an oncolytic virus, vsv m51 could replicate selectively and potently in tumor cells, but rarely in normal cells with competent type i ifn signaling. an ideal replication competent vaccine vector should possess a suitable balance between pathogenesis and capability to stimulate immunity effectively. both vsv-g 28 and vsv m51-g 28 have demonstrated significant attenuation compared to vsv xn2 or vsv m51, which are considered safe as vaccine vectors. however, we sought to determine which attenuated virus would work better in stimulating potent protective immunities that are critical for their clinical applications. to evaluate protective immunities stimulated by vsv-g 28 and vsv m51-g 28, animals were immunized with different doses of viruses and then challenged with a lethal dose of vsv xn2 , which is the "gold standard" to evaluate efficacy of a vsv vaccine. as shown in the current study, vsv-g 28 could stimulate more potent protective immunities than vsv m51-g 28. of note, vsv-g 28 treated animals inoculated with a low 10 3 pfu dose, no deaths or significant body weight losses were observed post-challenge with vsv xn2 . however, animals in vsv m51-g 28 treated group suffered serious body weight loss even when immunized with 10 4 pfu and some table 2 serum neutralizing antibody titers in mice against vsv indianna after inoculation with vsv-g 28 or vsv m51-g 28. animals were inoculated with 10 3 or 10 4 pfu of different vsvs with pbs as a control. sera were collected before and 21 days after inoculation. neutralizing antibody titers against vsv indiana were determined and expressed as the reciprocal of the highest dilution of antibody giving a 100% inhibition of cytopathic effect. '-': not detectable. animals died when the dose was as low as 10 3 pfu. the results correlated with neutralization antibody titers stimulated by different vsvs. neutralization antibodies titers in animals vaccinated with vsv-g 28 were shown to be much higher than those with vsv m51-g 28. therefore, although vsv m51-g 28 contained the double mutated m and g proteins and was a very safe viral vector, it was not as effective as vsv-g 28 in stimulating protective immunity, possibly due to its extreme attenuation in vivo. in summary, among different attenuated vsvs with mutated m or/and g proteins, recombinant vsv with only truncated g protein (vsv-g 28) indicated ideal balance between pathogenesis and capabilities in stimulating protective immune response and could be a promising vaccine vector. however, for the purpose of developing a vaccine candidate for the prevention of a vsv pandemic, these vaccine candidates would need to be evaluated in swine and cattle, which are the natural host of vsv, before its application in the field. in the current study, a novel recombinant vsv was constructed with mutations in both m ( m51) and g (g 28) proteins. for the first time, vsv with mutated m and/or g proteins (vsv m51, vsv-g 28, vsv m51-g 28) were compared to evaluate their potentials as vaccine vectors. the experimental conclusions included: (1) both g 28 and m51 contribute to the attenuation of vsv, however, g 28 is likely to play a more important role than m51. vsv m51-g 28 was determined to show the most significant attenuation in vitro. (2) virulence of recombinant vsvs with truncated g protein (vsv-g 28, vsv m51-g 28) significantly decreased compared with wild-type vsv or vsv m51. (3) vsv-g 28 could stimulate a more potent protective immune response than vsv m51-g 28 possibly due to the extreme attenuation of vsv m51-g 28. among different attenuated vsvs with mutated m and/or g proteins, recombinant vsv with only truncated g protein (vsv-g 28) displayed an ideal balance between pathogenesis and stimulation of a protective immune response that could be used as a promising vaccine vector. recombinant vesicular stomatitis viruses from dna efficient recovery of infectious vesicular stomatitis virus entirely from cdna clones foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles recombinant vesicular stomatitis (indiana) virus expressing new jersey and indiana glycoproteins induces neutralizing antibodies to each serotype in swine, a natural host properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses a single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (sars-cov) s protein results in the production of high levels of sars-cov-neutralizing antibodies evaluating replication-defective vesicular stomatitis virus as a vaccine vehicle vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates rhabdoviridae: the viruses and their replication the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host a proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with ww domains of cellular proteins: implications for viral budding modifications of the psap region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo vsv strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immunecompetent mice attenuated vesicular stomatitis viruses as vaccine vectors ns1 proteins of avian influenza a viruses can act as antagonists of the human alpha/beta nterferon response homozygous deletion of the alpha-and beta 1-interferon genes in human leukemia and derived cell lines influenza a virus lacking the ns1 gene replicates in interferon-deficient systems rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine development sensitivity of prostate tumors to wild type and m protein mutant vesicular stomatitis viruses effects of intravenously administered recombinant vesicular stomatitis virus (vsv(deltam51)) on multifocal and invasive gliomas impaired interferon signaling is a common immune defect in human cancer key: cord-292593-apdyaujt authors: coulter-mackie, marion; adler, richard; wilson, greame; dales, samuel title: in vivo and in vitro models of demyelinating diseases xii. persistence and expression of corona jhm vims functions in rn2-2 schwannoma cells during latency date: 1985-10-31 journal: virus research doi: 10.1016/0168-1702(85)90049-8 sha: doc_id: 292593 cord_uid: apdyaujt abstract the coronavirus jhmv persistently infects rat schwannoma cells rn2-2 at 32.5°c and enters a host-imposed reversible, latent state at 39.5°c. jhmv can remain up to 20 days in the latent state and about 14 days before the cultures lose the capacity to resume virus production upon return to 32.5°c. although persistently and latently infected rn2-2 cells display resistance to superinfection by a heterologous agent vsv, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. nevertheless, rn2-2 cells are competent to synthesize and release interferon when treated with the appropriate inducers. these observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the schwannoma cell. hybridization with virus-specific cdnas shows that all viral mrnas are present during latency and that viral mrnas are present in the polysomes of infected cells at 39.5°c. western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. these results the coronavirus jhmv persistently infects rat schwannoma cells rn2-2 at 325°c and enters a host-imposed reversible, latent state at 395°c. jhmv can remain up to 20 days in the latent state and about 14 days before the cultures lose the capacity to resume virus production upon return to 325°c. although persistently and latently infected rn2-2 cells display resistance to superinfection by a heterologous agent vsv, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. nevertheless, rn2-2 cells are competent to synthesize and release interferon when treated with the appropriate inducers. these observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the schwannoma cell. hybridization with virus-specific cdnas shows that all viral mrnas are present during latency and that viral mrnas are present in the polysomes of infected cells at 39.5"c. western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. these results suggest that despite the absence of production of infectious virus at 39s"c, there is active transcription and translation into virus-specified products. latent persistent infection, coronavirus, rat, schwannoma cell viruses of many types are associated with the etiology of neurological diseases in animals and man. among the rna agents the coronaviruses have sparked considerable interest in connection with demyelinating diseases of the cns. the murine agents of the mouse hepatitis virus (mhv) group, constituting several serotypes, have in particular been studied extensively in persistent infections in vitro (holmes and behnke, 1981; stohlman and weiner, 1978; lucas et al., 1977) and in vivo (knobler et al., 1981; hirano et al., 1980; nagashima et al., 1979; sorensen et al., 1980 sorensen et al., , 1982 sorensen et al., , 1984 . the epidemiological surveys which revealed that in some countries essentially 100% of the human population is seropositive (hovi et al.. 1979; hasony and macnaughton, 1982; macnaughton, 1982) attest to the ubiquity of these agents. in various studies we have described a model involving the persistent infection of rn2-2 rat schwannoma cells by the neurotropic coronavirus jhmv (lucas et al., 1977 (lucas et al., , 1978 . with this agent, virus production, measured by the quantity of extracellular infectious particles, occurs in a cyclical fashion when cultures are maintained at 32.5"c. elevation of temperature to a restrictive 39.5"c interrupts jhmv production. however, infectious particle formation may be resumed when the cultures are returned to the permissive 32.5"c. in these cell-virus systems the control on replication, related to alterations in the temperature, appears to be imposed by the host cell because thermosensitivity of either virus is insufficient to account for the observed temperature-related restriction of virus production, termed hereafter latency. on the assumption that during prolonged restriction imposed by the host rn2-2 cell, latent jhmv are somehow perpetuated, we focussed our attention on the nature of the incomplete virus expression, particularly the detection of viral genomes, mrna and proteins. data from studies presented here reveal that both viral mrna and protein are expressed during latency. the source and characterization of the rat schwannoma cell line, rn2-2, and mouse l-2 cells has been previously reported (lucas et al., 1977 (lucas et al., , 1978 . primary rat embryo cells (re) were derived from 15-day embryos of the wistar rat. cells were released from tissues by mincing and dispersal into phosphate-buffered saline, containing 22 mm glucose and 0.25% trypsin, employing agitation at 37°c for 15 min. the monodisperse re cells and continuous lines were grown in nutrient medium (nm) consisting of eagle's minimal essential medium supplemented with 5 or 10% fetal bovine serum (fbs). the origin and propagation methodology for jhmv has been published (lucas et al., 1978) . virus titres, expressed as plaque-forming units per millilitre (pfd/ml) were measured by assay on l-2 cells for jhmv, according to lucas et al. (978) . in order to determine the fraction of cells yielding pfu in the persistently or latently infected rn2-2 cultures, the monolayers were trypsinized, free cells counted and then plated at 2-fold serial dilutions onto monolayers of the indicator l-2 cells, in 96-well microtitre plates. the co-cultures were incubated at 32.5"c for 4-6 days, then fixed with formaldehyde and stained with crystal violet. the minimum number of test cells added that were required to cause a cytopathic effect (cpe), i.e. the end point, became a measure of the frequency of infectious centers in the population of the culture. the initiation of persistent or latent infections by jhmv in rn2-2 cells has been described (lucas et al., 1978) . inocula are millipore-filtered before adding to cells (coulter-mackie et al., 1984) . interference with vesicular stomatitis virus (vsv) production by jhmv was tested in persistently or latently infected rn-2 cells using as the controls, uninfected rn2-2 cells. for this purpose test monolayers, in 35 mm dishes, were inoculated with lo2 pfu of vsv, incubated for 24 h, then fixed and stained for plaque counting. the efficacy of induction of an antiviral state was measured as interference with vsv replication. to produce an antiviral state, some uninfected cells were pre-incubated with nm containing o-20 pg/ml poly(i) : poly(c) (sigma) for 14 h. the medium was removed, cells were washed and then infected with vsv, as above. specifically the sampled medium was adjusted to ph 2 with 1 n hcl, kept for 48 h at 4°c then neutralized and assayed for content of interferon. two-fold serial dilutions of the test medium were added to rn2-2 or re cells in 96-well microtitre plates. after 24 h exposure at 37°c the medium was removed, cells were washed with pbs, then challenged with vsv. seven 600 cm2 culture plates (gibco) of rn2-2 cells were infected with jhmv at a m.o.i. of 1. five plates were maintained at 39.5"c and two at 32.5"c. at 24 h intervals titrations were made for virus production. cultures sampled at intervals were extracted for total rna. using guanidine-hcl (strohman et al., 1977) . purified rnas were denatured with glyoxal and electrophoresed on agarose gels (mcmaster and carmichael. 1977) six 150-cm2 culture flasks of rn2-2 cells were infected with millipore-filtered jhmv. three of these were maintained at 32.5"c and the other three continuously at 39.5"c. 9 days post-infection (pi) cells at 32.5"c were yielding virus at ca. 10' pfu/ml and those at 39.5"c at 0 pfu/ml. ['h]uridine (30 ci/mmol, new england nuclear) was added to a concentration of 2 yci/ml and cells were incubated a further 16 h. cell extracts containing polyribosomes (polysomes) were prepared according to gielkens et al. (1971) . cells which had been treated with 50 pg/ml cycloheximide for 20 min were trypsinized, then lysed in np40. the extract was layered on 20%39.6%' isokinetic gradients and centrifuged lo5 x g for 70 min. then the gradient was divided into aliquots and the labelled rna measured in a scintillation counter. the polysome-rich fractions were pooled, concentrated by ethanol precipitation and the pellets formed were taken up in a small volume of water. in preparation for dot-blotting analysis (white and bancroft. 1982) the solution was made 7% with respect to formaldehyde and 10% with respect to ssc giving a total volume of 0.5 ml. samples were heated 60 min at 50°c and then applied to nitrocellulose (biorad). using a schleicher and schuell 'minifold' filtration apparatus. nitrocellulose blots were hybridized with [j2p]cdna (jhmv), washed and subjected to autoradiography. preparation of the cdna and hybridization conditions have all been described (sorensen et al., 1984; coulter-mackie et al., 1980) . extracts of both uninfected and infected rna-2 cell cultures were prepared by washing twice with ice-cold phosphate-buffered saline (pbs), followed by solubilization for 10 min at 37°c in lysis buffer: 0.15 m tris-hcl, ph 8.8/10% glycerol/5% 2-mercaptoethanol/2% sodium dodecyl sulfate (sds). proteins were separated by sds-polyacrylamide gel electrophoresis (sds-page) in 10.5% acrylamide gels (laemmli, 1970) . replicas were prepared by electrophoretic transfer for 18 h at 6 v/cm onto 0.2 pm nitrocellulose filters (schleicher and schuell). the replicas were cut into strips, placed in pbs + 0.05% tween 20 for 1 h at 25°c and incubated for 2 h at 25°c with murine monoclonal antibodies (kindly supplied by dr. michael buchmeier, scripps clinic and research foundation, la jolla), according to towbin et al. (1979) . the blots were washed six times in pbs-tween 20, and rabbit anti-mouse kappa chains (miles laboratories) were added to serve as links between jhmv polypeptides and protein a. subsequently the strips were washed six times in pbs-tween 20, reacted for 2 h at 25°c with '251-labelled protein a, specific activity 5 pci/pg (new england nuclear) and finally washed six times in pbs-tween 20, dried, and exposed to x-ray film to detect radioactive bands by autoradiography. the phenomenon of coronavirus persistence at 32.5"c and profound suppression of virus production at 39.5"c in rn2-2 cells, and other continuous rat cell lines, is quite striking (lucas et al., 1977; 1978) . similar cell-virus interactions were shown, more recently, to occur in primary rat neural (sorensen et al., 1984; beushausen and dales, 1985) and embryonic re cells (m. coulter-mackie, unpublished), indicating that in appropriately selected cells, temperature restriction on virus replication is under host control. to ascertain the extent to which jhmv can be maintained latently at 39.5"c, a long-term study of events prevailing at the elevated temperature was undertaken. at intervals of 3-4 days, prior to change of nm, the extracellular virus titre was determined. at weekly intervals cells in monolayers were released by trypsinization, transferred into new culture flasks and perpetuated at 39.5"c. the fraction of cells which could function as virus yielders was determined by an infectious centre assay. the duration for which the latently infected cells maintained capacity to resume virus production at 32.5"c was also determined. resumption of virus production after temperature shift down from 39.5"c was usually detectable within 445 days at 32.5"c. under coculture conditions with indicator l-2 cells, cytopathic effects (cpe) due to jhmv became evident within 1-3 days after return to 32.5"c. the results of the infectious centre assays, summarized in fig. 1 , revealed that there was a progressive decrease, with time, in the fraction of infectious centres. rn2-2 cultures persistently infected with jhmv at 32.5"c usually contained o.l-10% virus yielding cells during the course of a well-established infection. since upon infection of rn2-2 cultures with jhmv at 39.5"c and maintenance in a state of latency at that temperature for several days, the cells commenced yielding pfu upon shift down to 32.5"c (lucas et al., 1978) , these data suggest that penetration and eclipse of the virus inoculum must have occurred normally at 39.5"c and restriction most probably developed at a subsequent stage. of jhmv in rn2-2 cells. infectious centres assays, as described in materials and methods, were carried out at weekly intervals on samples from cultures at 32s"c (0) or 39s"c (0). the data, plotted semiiogarith~cal~y, indicate a decline to zero (arrow), at the time infectious centres were no longer detectable. in the data shown, the experiment was initiated 28 days pi, i.e. with a well-established persistent infection. although infectious centres were detectable for about 20 days at 39.5"c the capacity of latently infected rn2-2 cultures to resume virus production, upon temperature shift down to 32.5'c, was retained for only about 14 days. apparently the infectious centres assay is the more sensitive measure of potential infectiousness. incubation of rn2-2 cells at 32s"c, 37"c, or 395°c for 24-48 h prior to infection had no effect on the outcome of the infection, indicating that the observed inhibition of jhmv replication does not result from induction of factors in the uninfected cells when they are cultured at the elevated temperature. one approach towards rescue of jhmv from latently infected rn2-2 cells was by means of co-culture with the indicator line l-2, host cells which do not restrict virus production at 395°c. upon co-cultivation for l-3 days cell-cell fusion became evident, typical of that seen during lytic infection of l-2 cells. these observations revealed the presence of latent jhmv which could be transferred to indicator cells upon close cell-cell contact and therein established a lytic infection. to test whether some factor such as interferon (ifn), or an interferon-like effect, prevailed during persistence or latency in rn2-2 cells, jhmv-infected cultures were superinfected with vsv at either 32.5 or 395°c. such cells were, indeed, resistant to challenge with vsv showing 100% inhibition of vsv inoculated at the m.o.i. of 1. the procedure is described in materials and methods. cells were inoculated with 2 x 10 2 pfu of vsv and checked for plaque counts 24 h later. results are given as the percent reduction in vsv titer compared to untreated controls. control cells gave plaque counts in the range of 180-200 on re cells and 150-175 on rn2-2 cells. a uninfected cells were treated with 10 pg/ml poly (1) : poly(c). ' poly (1): poly(c)-containing medium, collected from cells being tested for the cell-associated effect, was rnase treated and tested on fresh rn2-2 and re cells for its ifn inductive effects. these results revealed that although only a small fraction of the cells in a population were scored as positive for jhmv, the ongoing infectious state of the culture was sufficient to inhibit totally the replication of vsv. this finding suggests that persistent or latent infections of only a few cells were adequate to protect the entire culture against vsv. the above results led us to initiate tests for the presence of antiviral material i.e. ifn. for this purpose, 'conditioned' nm from latently or persistently infected rn2-2 cells was used to treat uninfected rn2-2 cells prior to inoculation with vsv, as described in materials and methods. there was no significant reduction in the titer of vsv produced. nor was evidence found for presence of extracellular mediators in 'conditioned' nm from which the protein had been concentrated by procedures used to concentrate rat ifn, described by schellekens et al. (1980) . these findings indicate that, within the limits of detection of our assay, persistently or latently infected schwannoma cells were not producing any soluble interferon or ifn-like material. failure to detect ifn during persistence and latency led us to investigate whether the schwannoma line, like primate vero cells (desmyter et al., 1968) is deficient for ifn inducibility, as defined by lockart (1973) . the analyses involved both the ability of test cells to become resistant to vsv and to produce a transferable, soluble factor which conferred resistance against vsv. our results, summarized in table 1 , showed that primary rat embryo (re) cells were highly sensitive to the interferon inducer poly(1) : poly(c), both with respect to acquisition of resistance to vsv and production of a soluble antiviral material. by comparison, rn2-2 cells were affected to a lesser extent by this inducer. however, exposure of either re or rn2-2 cells to uv-inactivated reovirus type 3 produced maximal responses within 24 h in both cell types and some ifn was already detectable by 6-7 h after treatment. following addition of inactivated reovirus, equivalent to 10-1000 pfu/cell of live virus, the same amount of ifn in the nm was produced by either re or rn2-2 cells, about 1.500 units/ml by comparison with a standard ifn preparation. this demonstrated that both the rat cell types tested had equal responsiveness to induction of ifn by reovirus. from this one can conclude that rn2-2 cells are not lacking in capacity to make ifn. despite absence of detectable ifn in jhmv-infected culture medium, the rn2-2 cells were resistant to superinfection with vsv. this led us to test the influence of exogenously added ifn by exposing persistently or latently infected cultures to purified rat ifn, characterized as the (y type (lee biomolecular). preliminary tests revealed that addition of 50 units/ml of this ifn reduced by over 75% the titer of vw in previously uninfected rn2-2 cells. on the other hand, treatment of this line, with 20-200 units/ml of the rat ifn, at the time the cells were already undergoing persistent infections with jhmv, failed to decrease significantly the yields of jhmv. therefore, jhmv persistence in this system does not appear to be sensitive to or under the control of ifn. 'northern' transfer hybridization with isotopically labelled cdna probes, specific for jhmv was conducted using extracts of rna from cultures maintained after infection at either 32.5 or 39.5"c. bands were evident in the autoradiograms at positions corresponding generally to those associated with mrnas of jhmv (fig. 2) . the number and approximate m,'s of the mrna bands (l-7) agreed with findings from lytic infection of l cells, as reported by cheley et al. (1981b) , except that the band related to the 19s nucleocapsid mrna (mrna 7) was evident at a position of slightly lower m, than anticipated. this was most probably due to a displacement by the presence of relatively much more abundant 18s rrna in the vicinity, which could have produced a distortion of the migration of the viral mrna in the agarose gels. it was noted that mrna 6, which has an approximate molecular weight of 1.15 x lo", showed a secondary faint band of slightly lower molecular weight. the lower molecular weight band predominated when jhmv was grown lytically in l-2 cells (data not shown). these findings are of interest in light of those of taguchi et al. (1985) who observed alterations in the molecular weights of mrnas 2 and 3 in brain-derived vs. culture cell-derived material. throughout persistent infection of rn2-2 cells, at 32.5"c. all species of jhmv mrna were detected, as illustrated with a 4-day pi sample in channel a of fig. 2 . judging by the unchanged intensity of the bands with time of sampling, the quantity of mrna did not vary appreciably. by contrast, during latency at 39.5"c, the intensity of the bands decreased with time elapsing pi as evident in channels b-e. thus, by day 4 pi at the elevated temperature, the bands became faint. an extract of rna from a culture, kept in a state of latency for 11 days, apparently contained 4-!i-7--28s *l es fig. 2 . 'northern' transfer and cdna hybridization to rna extracted from latently or persistently jhmv infected rn2-2 cells. data after incubation at 32.5"c for 4 days pi are in channel a; data from infection at 39.5"c for 4, 3, 2, 1 or 11 days pi in channels b-f, respectively. material in f was from a separate experiment and involved a 2 x longer exposure time for the autoradiogram. rna extracted from uninfected cells in channel g. on the left-hand side are indicated the m,'s of jhmv mrnas 1 to 7, which are respectively 3.7 x 10 x 106, 3.4~ 106, 3.1 x 106, 1.6 x 106, 1.45 x 106, 1.15 x 10' and 6.3 x 10'. location of the 28s and 18s ribosomal rnas is shown by arrows on the right side of the figure. mrna corresponding to the nucleocapsid gene. these results suggested that early during latency all or most of the jhmv mrnas were being expressed but later than 4 days pi the nucleocapsid mrna, the most abundant viral mrna (stern and kennedy, 1980) was the only mrna detectable by these means. the data suggest that transcription at the restrictive temperature continued but the abundance of the mrnas decreased rapidly within a few days. the results from cdna probing of transcripts are consistent with the observed decline in the number of infectious centers which became evident as the duration of latency was prolonged (see fig. 1 ). to ascertain whether active translation was underway at 39.5"c, polysomes from infected rn2 cells were isolated, fractionated and subjected to hybridization with jhmv-specific [ 32p]cdna and autoradiography, as described in materials and methods. the autoradiograms, shown in fig. 3 , indicated that mrna specified by sucrose isokinetic gradients and centrifuged at 10' x g for 70 min. the gradients were fractionated and acid insoluble cpm determined. fractions from 3 major areas of each gradient were pooled (a, b and c), precipitated with ethanol and used for 'dot-blotting', as described in the materials and methods. the incubation temperatures for the persistently or latently infected rn2-2 cells, from which the extracts were made, are indicated. the 'dot-blot' hybridization autoradiograms of the pooled gradient fractions are shown above the gradient profiles. the columns designated a, b and c correspond to similarly identified pooled fractions, shown below. the lower dot in each panel represents l/10 of the total material in the pooled fractions. the upper dot is a l/10 dilution of material present in the lower dot. the centre panel of the autoradiogram shows hybridization results with polysome extracts from uninfected rn2-2 cells. jhmv was present in the gradient fractions enriched for polysomes, isolated from cells kept at either 32.5 or 39.5"c for 9 days pi. this finding suggests that active translation of coronavirus messengers was also taking place at the restrictive temperature, in the absence of infectious virion formation. since it was apparent from work with polyribosomes that some jhmv transcription occurred during latency at 39.s°c, analyses were made to ascertain whether presence of viral mrna could be related to translation into viral poi~~ptide(s). as already mentioned, incubation of infected rn2-2 cells at the permissive 32s"c was associated with only minimal cytopathology, implying that detection of viral polypeptide might be different, especially during latency at 39s"c. to maximize the sensitivity of detection, virus related antigens were identified by immunob~otting and autoradiography. for this purpose, infected rn2-2 cells, used as controls, were cultured at the permissive or restrictive temperatures. samples were removed at intervals and extracts prepared from them, then subjected to sds-page, immunoblotting with hybridoma antibodies and autoradiography. the results from the permissive conditions, after tagging with anti-nu~leocapsid antibodies, are presented in fig. 4 . it is evident from channel b, representing material sampled 12 h after inoculation, that there was a significant quantity of material in a band at the 56000 da (56k) nucleocapsid position, corresponding to the m, of primary product of viral nucleocapsid (cheley and anderson, 1981a) . the other bands with apparent m,'s 31k and 33 k, evident in these gels, were presumably irrelevant and artifactual, since they occurred in other channels, including those representing uninfected controls whenever the anti-mouse kappa chain antibody was utilized as a linker. lanes c-i of fig. 4 represent immunoblots of extracts from, respectively, uninfected rn2-2 cells kept at 32.5"c those sampled 60 min after infection (0 h) at 32.5"c or respectively at 12, 24, 48, 72 h and 7 days post-inoculation. in samples prepared 60 min after inoculation the antibodies could identify a predominant polypeptide band of 50k and a minor one of 48k. in samples taken 12 h after inoculation the antigen(s) recognized by the hybridoma was 56k and 50k, as evident in lane e. previous work by cheley and anderson (1981a) , confirmed by our own studies, established by means of pulse-chase labelling experiments that a precursor-product relationship exists between the 56k and 5ok polypeptides. prominent 56k and 50k antigens were detected also in cell extracts for 7 days or longer after inoculation, revealing that production of nucleocapsid protein was continuous throughout the course of the persistent infection. there was therefore, good correspondence between data on jhmv transcription (figs. 2 and 3) translation and formation of infectious jhmv. by contrast, identical immunoblotting utilizing extracts of latently infected rn2-2 cells, maintained at 395°c gave somewhat different results. as evident in channel b of fig. 5 , in the infected l-2 cells, used as controls, the predominating antigen was 56k. with rn2-2 cells sampled at 0 h (60 min after inoculation), the predominant form of nucleocapsid species was the 50k antigen (fig. 5, channel d) . similar antigenic bands were identified in the 12 h samples (fig. 5 , channel e). it is. however, not clear from these data alone whether the 50k band material is nascent antigen or the residual antigen introduced with the inoculum. judging by the bands corresponding to the nucleocapsid, the antigen which accumulated at 39.5"c, when infection was extended beyond 12 h, was the 56k precursor polypeptide (channels f-i of fig. 5 ). it is, therefore, most probable that the 50k polypeptide evident in channel d was an intracellular cleavage product of nascent 56k since, with time, the 50k component decreased at a relatively faster rate than the 56k antigen. it is very significant that during incubation at the restrictive temperature for as long as 7 days and beyond, when rn2-2 cells had multiplied through 6-7 generations, the 56k antigen was readily detectable (channel i), implying that during latency, in the absence of infectious virus production, translation into jhmv nucleocapsid protein continued. this finding is consistent with the presence of jhmv-specified mrna in the polyribosomes isolated from rn2-2 cells, latently infected for 9 days (fig. 3) . probing for the expression of the other structural components, the e, and e, glycoproteins, of apparent m, 21k and isok, respectively, was also undertaken. employing anti-e, and anti-e, hybridoma antibodies of collins et al. (1982) for immunoblotting, it was found that at the permissive temperature both antigens were expressed in rn2-2 cells, but during latency at 39.5"c neither glycopeptide was detectable (data not shown). these findings suggest that the temperature-related inhibition of infectious virus formation might be due either to the selective suppression of the synthesis or rapid turnover of jhmv envelope components, while production of the nucleocapsid polypeptide is much less affected. on the other hand, the normally greater abundance of the nucleocapsid polypeptide than of the other structural components could account for our inability to detect e, or ez in rn2-2 cells at 39.5"c. the present study was feasible because it is possible upon infection of rat schwannoma cells rn2-2 with jhmv, to establish rapid and reproducible persistence or latency, influenced by elevated temperature, as demonstrated in an earlier report by lucas et al. (1978) . thus, when infections are initiated at 39.5"c the virus is able to establish itself without formation of infectious virions. the temperature restriction on the replication process is most probably controlled by a host-imposed function and depends on the continued presence of the virus in state of latency, since susceptibility to reinfection is restored at 32.5"c, after rn2-2 cells become 'cured' of the infection. the prolonged latency of jhmv under restrictive conditions is remarkable. in comparison, measles virus which also establishes a persistent infection in rn2-2 cells (lucas et al., 1978) has shown a latency period of up to 70 days (coulter-mackie and dales, unpublished observations). the duration of latency with jhmv during which reactivation remains possible is shorter, perhaps only 14-20 days. the difference between the two viruses is probably related to the proportion of cells in a culture which carry viral information, the fraction involved with jhmv persistence being much lower than with measles virus persistence. the conversion of jhmv infection from the persistent into latent states upon temperature elevation to 39s"c, and the prolonged latency which can occur in the in vitro cultures, could have direct bearing on understanding the infectious process during jhmv infection of the rat central nervous system (cns). since symptom-free rats. surviving an initial intracerebral infection with jhmv, can be caused, following treatment with the immunosuppressive agent. cyclophosphamide, to develop a chronic. demyelinating disease (sorensen et al., 1982) . it is quite possible that this virus can be perpetuated in a covert form within the cns. consistent with this view is the finding that rats which remain asymptomatic for as long as 150 days after inoculation may harbour jhmv rna in the cns (sorensen et al.. 1984 ). in the present study several approaches were taken towards an understanding of the nature of the block in virus production at the elevated temperature. as previously discussed by lucas et al. (1978) , involvement of defective-interfering particles or development of thermosensitive virus variants was not demonstrable and, therefore, is unlikely to account for the inhibition observed. host factors might be responsible for the observed virus suppression. one possible candidate for the host factor in question is ifn. it is' well known that during chronic in vitro infections with neurotropic agents, such as rabies virus, there is an inverse correlation between the cycling virus titre and ifn levels in the medium (wiktor and clark, 1972) . it may also be significant in the present context that ifn activity can be enhanced at elevated temperature (heron and berg, 1978) . thus, in our system it was postulated that ifn or interferon-like substance might influence the control of the persistence at 325°c and suppression of infectious particle formation at 39.5"c. although in the present study the development of resistance to infection of rn2-2 cells by a heterologous virus. vsv. could be demonstrated during persistent and latent infections with jhmv and likewise with measles virus (coulter-mackie and dales, unpublished observations), no evidence was obtained to demonstrate that such persistently or latently infected cells produce ifn or an ifn-like substance. these two viruses differ in that measles virus is known to be an ifn inducer (demaeyer and enders, 1961: mckimm-breschkin and rapp, 1981) while jhmv probably does not induce ifn in cultured cells. although replication of this coronavirus is sensitive to exogenous ifn (garlinghouse et al., 1984) . however, since these schwannoma cells are inducible for ifn with uv-inactivated reovirus and can respond to exogenously added ifn. yet continue to produce jhmv at a normal rate after the addition of purified rat ifn to persistently infected rn2-2 cultures. it is questionable whether ifn exercises any control in this system or that involving rat neural cells in general. therefore, the interference with vsv replication during latency or persistence remains unexplained. prqbing for jhmv-specific rna by means of 'northern' transfer indicated that transcription can occur at both 32.5 or 39.5"c. while at 39.5"c the mrna specifying the nucleocapsid was the predominant species and was still evident on the in vivo and in vitro models of demyelinating disease xi. tropism and differentiation regulate the infectious process of coronaviruses in primary explants of the rat cns to the other viral functions could be detected only during the first few days post-inoculation.however, the corresponding immunoblots made on extracts from latently infected cells could detect only the 56k nucleocapsid protein, suggesting that preferential translation of the nucleocapsid mrna occurs in the repressed state. the situation regarding the other major jhmv structural polypeptides e, and e, remains unresolved. while these glycoproteins were expressed normally at the permissive temperature, we were unable to detect their presence during latency, despite the presence of mrna species corresponding to e, and e, during the initial 2-3 days of latency. these data suggest that infection at the elevated temperature may lead to either a differential inhibition of the synthesis of envelope polypeptides, or their rapid turnover, or both. the western blot analysis also revealed a processing step connected with the nucleocapsid protein, which is blocked during latency. the nucleocapsid is initially produced as the 56k form and integrated into the virus. soon after infection, however, the nucleocapsid material of the inoculum exists as a 50k molecular weight polypeptide (figs. 4 and 5) . this precursor-product relationship has been documented by cheley and anderson (1981a) . both sizes of this antigen were found to be phosphorylated (our data, not shown, and siddel et al., 1983) . the 50k proteins found in infected rn2-2 cells incubated at 32.5"c are likely. therefore, to represent the product of processing which may occur during cell-to-cell spread of progeny virus. upon incubation of rn2-2 cells at 395°c assembly and/or spread of the virus progeny are probably inhibited. this is consistent with our preliminary observations (adler et al., unpublished) , which reveal that compounds interfering with jhmv entry into l-2 cells also affect processing of 56k nucleocapsid of inoculum virions.from the above it is clearly evident that in latently infected rn2-2 cells the viral genomes are not dormant and merely segregated to daughter cells but are instead, active in transcription and translation. it remains to be shown, first of all, whether genome duplication is reduced or stopped. secondly, whether the expression of some functions is specifically reduced during latency and thirdly, why the production of infectious progeny ceases. 45. 2055213. schellekens, h., dewilde. g.a. and weimar. w. (1980) production and initial characterization of rat interferon. j. gen. virol. 46, 2433247. siddell. s.. wege, h. and ter meulen. v. (1983) the biology of coronaviruses.j. gen. virol. 64, 761-776. sorensen, 0.. percy, d. and dales, s. (1980) in vivo and in vitro models of demyelinating disease iii. jhm virus infection of rats. arch. neurol. 37, 478-4x4. sorensen. 0.. dugre, r.. percy. d. and dales. s. (1982) key: cord-275348-jna496x7 authors: kapadia, sagar u.; simon, ian d.; rose, john k. title: sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector date: 2008-06-20 journal: virology doi: 10.1016/j.virol.2008.03.002 sha: doc_id: 275348 cord_uid: jna496x7 a sars vaccine based on a live-attenuated vesicular stomatitis virus (vsv) recombinant expressing the sars-cov s protein provides long-term protection of immunized mice from sars-cov infection (kapadia, s.u., rose, j. k., lamirande, e., vogel, l., subbarao, k., roberts, a., 2005. long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine. virology 340(2), 174–82.). because it is difficult to obtain regulatory approval of vaccine based on live viruses, we constructed a replication-defective single-cycle vsv vector in which we replaced the vsv glycoprotein (g) gene with the sars-cov s gene. the virus was only able to infect cells when pseudotyped with the vsv g protein. we measured the effectiveness of immunization with the single-cycle vaccine in mice. we found that the vaccine given intramuscularly induced a neutralizing antibody response to sars-cov that was approximately ten-fold greater than that required for the protection from sars-cov infection, and significantly greater than that generated by the replication-competent vector expressing sars-cov s protein given by the same route. our results, along with earlier studies showing potent induction of t-cell responses by single-cycle vectors, indicate that these vectors are excellent alternatives to live-attenuated vsv. sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector introduction sars (severe acute respiratory syndrome) emerged in the fall of 2002 in china but soon caught the world's attention as it quickly spread to 28 countries. by the end of 2003 the world health organization reported over 8000 probable cases of sars, a fifth of which occurred in health care workers. the overall fatality rate was 9.6%, but in people over the age of 60, the rate exceeded 50%. (http://www.who. int/csr/sars/en/whoconsensus.pdf; peiris et al., 2004) the etiological agent was quickly identified as a coronavirus (cov) ksiazek et al., 2003) , and the 30 kb genome sequence revealed a common coronavirus genome organization (marra et al., 2003; rota et al., 2003) . six major open reading frames were identified. of those, four encoded the major structural proteins: spike (s), membrane (m), nucleocapsid (n) and envelope (e). m, n and e are involved in viral assembly and budding. s, the major glycoprotein, binds the cellular receptor, ace 2 , and mediates entry by a class i viral fusion mechanism (bosch et al., 2003) . there have been no reported cases of sars since 2004; however sources of the sars-cov still exist. animal carriers of the virus including himalayan palm civets, raccoon dogs and bats have been identified (guan et al., 2003; lau et al., 2005; li et al., 2005) . several cases of laboratory-acquired sars have also been reported. because sars-cov has not been eradicated, there is still a potential for human infections. a sars vaccine may be important in controlling future outbreaks. several experimental vaccines have been constructed and tested. these include dna vaccines, protein subunit vaccines, inactivated sars-cov vaccine and recombinant viral vaccines (gillim-ross and subbarao, 2006) . the sars-cov s glycoprotein has been used as the antigen in the development of most of these sars vaccines because it is the target of virus neutralizing antibody. we previously reported the development of an experimental vsv-based sars vaccine. vsv (vesicular stomatitis virus) is a negative strand rna virus that belongs to virus family rhabdoviridae (kapadia et al., 2005) . attenuated vectors derived from vsv have been used extensively as experimental vaccine candidates (daddario-dicaprio et al., 2006a,b; egan et al., 2004; geisbert et al., 2005; jones et al., 2005; kahn et al., 2001; natuk et al., 2006; palin et al., 2007; ramsburg et al., 2004; reuter et al., 2002; roberts et al., 1999 roberts et al., , 1998 roberts et al., , 2004 rose et al., 2001; schlereth et al., 2003 schlereth et al., , 2000 . they induce strong antibody and cellular immune responses, and with the exception of some rural populations in central and south america, there is negligible seropositivity to vsv in the human population (reif et al., 1987) making them attractive candidates for human vaccination. for populations with pre-existing immunity to vsv, nonendemic vsv serotype vectors can be used. vsv also grows to high titers in cell lines approved for vaccine production. in our initial study (kapadia et al., 2005) we showed that a vsv recombinant expressing the sars-cov s protein was capable of generating neutralizing antibodies against sars-cov in mice. furthermore, the immunized mice were protected from a sars-cov challenge. we also showed that a humoral response was sufficient for protection. in the current study we generated and tested the effectiveness of a vsv recombinant that is capable of undergoing only one round of infection because it lacks the gene encoding the vsv glycoprotein (g). use of such a replication-deficient vector would overcome the complex regulatory issues related to approval of live-virus vectors for use in humans. however, production of such vectors would require a qualification of a cell-line that expresses vsv g or some plasmid dna based complementation. furthermore, a single-cycle viral vaccine would alleviate concerns over potential risks related to the use of live viral vectors in individuals with weakened immune systems. in order to evaluate this vector as a sars vaccine candidate, we also developed a sars-cov neutralization assay using a pseudotyped vsv recombinant expressing a green fluorescent protein. in order to recover a single-cycle vsv recombinant encoding the s protein of sars-cov, the vsv glycoprotein (g) gene in a plasmid expressing the vsv anti-genome was replaced with a gene encoding sars-cov s (fig. 1a) . the resulting plasmid, pvsvδg-sars s, was used to recover a virus, vsvδg-s on bhk-21 cells expressing vsv g. because g is required for virus entry, vsv recombinants lacking the g gene must be complemented with g in order to produce infectious particles. viruses the vsvδg-egfp1 lacks the vsv g gene and has an egfp gene inserted into the first position of the genome. the rna sequences are shown in the (+) anti-genomic sense. (c) bhk-21 cells were infected with either vsvδg-s or wt vsv. cells were fixed, and sars-cov s was visualized by indirect immunofluorescence. the fluorescence images are shown on the left, and differential interference contrast (dic) images are shown on the right. (d) lysates of metabolically labeled bhk-21 cells infected with wt vsv (lanes 1 and 2), vsv-s (lanes 3 and 4) or vsvδg-s (lanes 5 and 6) were analyzed by sds-page. the lysates were also treated with pngase f to remove n-linked glycans from proteins (lanes 2, 4, and 6). complemented with g can infect cells for a single cycle, but do not propagate further in the absence of a complementing g protein. when vsvδg-s was used to infect bhk-21 cells, we observed only single infected cells and no virus spread, consistent with the absence of an encoded vsv g protein. to determine if the s protein was expressed by this recombinant, we examined cells using indirect immunofluorescence microscopy. bhk-21 cells were infected with vsvδg-s or wild type (wt) vsv, fixed, and then incubated with serum from a sars-cov-infected mouse. a secondary, alexa fluor 488-conjugated anti-mouse antibody was used for visualization by confocal microscopy (fig. 1c) . we found that the sars-cov s protein was expressed on the cell surface as indicated by the strong surface fluorescence signal visible in cells infected with vsvδg-s but not on control cells infected with wt vsv. to evaluate viral protein expression further, we infected bhk-21 cells with wt vsv, vsv-s (kapadia et al., 2005) or vsvδg-s and metabolically labeled cells with [ 35 s]-methionine. lysates of radiolabeled cells were analyzed by sds-page. because vsv infection shuts off host protein synthesis, the five vsv proteins l, g, n, p and m are readily seen without immunoprecipitation (fig. 1d , lane 1). vsv-sinfected cells expressed the sars-cov s protein in addition to the five vsv proteins (fig. 1d , lane 3). vsvδg-s-infected cells expressed s and all of the vsv proteins except g (fig. 1d , lane 5). because s is a highly glycosylated protein, we also treated the lysates with pngase f to remove glycans in order to further characterize s. after digestion, s migrated faster on the gel (fig. 1d , lanes 4 and 6), a change consistent with removal of the predicted 17 glycans (kapadia et al., 2005) . because a low level of s protein of sars-cov is incorporated into vsv particles (data not shown), it is possible that the s protein might mediate infection in the absence of g. to determine if this s protein could mediate infection of vsvδg-s, we infected vero e6 cells [cells that express the sars-cov receptor, ace2 li et al., 2003) ] with g-pseudotyped vsvδg-s. using an immunofluorescence microscopy assay for observing vsv n protein expression, we saw single infected cells after 6 h but observed no infected cells after 24 h despite a near confluent monolayer of live vero e6 cells. this result indicates that vsvδg-s is not capable of a second round of infection. additionally, we passaged vsvδg-s through bhk-21 cells to yield progeny lacking vsv g and attempted to infect vero e6 cells with this virus stock. even though we could detect s protein in these noncomplemented virions by western blot, we did not observe any infection of vero e6 cells with these particles. these results indicated that the s protein present on the virion was not capable of mediating vsv entry in tissue culture. this is consistent with reported results showing that the full-length s protein, as is encoded in vsvδg-s, is not capable of mediating infection of pseudotyped vsv (fukushi et al., 2005) . to determine if vsvδg-s is able to replicate in vivo without vsv g, we inoculated mice intramuscularly (i.m.) with non-complemented vsvδg-s. if there were any infection by this virus, we anticipated that there might be detectable immune responses to the s protein. we measured sars-cov neutralizing antibody response as a measure of replication. to control for possible immune responses to s protein on the surface of particles in the inoculum, we also administered uvinactivated, non-complemented vsvδg-s. as additional controls we immunized two groups of mice with either g-complemented vsvδg-s or uv-inactivated, g-complemented vsvδg-s. a dose of 5 × 10 5 pfu (plaque forming units) of the g-complemented vsvδg-s was used. an equivalent particle dose of the non-complemented virus was assessed from the amount of n protein in the virus preparation as determined by western blot. one month after inoculation, serum was collected from each animal and the sars-cov neutralization titers were determined. only the g-complemented vsvδg-s-inoculated animals generated any measurable neutralizing titers to sars-cov ( fig. 2 ). they averaged 1:128. the animals in the remaining groups including those inoculated with non-complemented vsvδg-s made no measurable neutralizing antibody response (even at an antibody dilution of 1:5) indicating that significant replication was not occurring. uv-inactivated g-complemented vsvδg-s did not induce a neutralizing antibody response, indicating that one round of replication is essential for a response. we also assessed the immune responses to the vsv vector in these animals. we used the serum from each animal to stain vsv-infected cells and observed vsv n expression by immunofluorescence microscopy. all animals immunized with live g-complemented vsvδg-s had an antibody response to n, while animals inoculated with noncomplemented vsvδg-s had no detectable response to vsv. this further supports the idea that vsvδg-s is not infectious in animals without vsv g. in order to test the potential of our single-cycle vsv recombinant as a sars vaccine, we conducted a study including five groups of mice. the first group included three control mice that received wt vsv intranasally (i.n.). the second group of three mice was inoculated with wt vsv i.m. the third group of six mice was immunized with vsv-s administered i.n., while the fourth group of six mice received vsv-s i.m. the last group of five mice was vaccinated with vsvδg-s i.m. a single vaccine dose of 5 × 10 5 pfu was administered. serum was collected from all mice at 5, 9 and 13 weeks post-immunization. to verify that all mice had been infected with the vectors, we measured vsv neutralizing antibody titers in the serum of individual mice at five weeks post-infection (fig. 3) . vsv g protein is the target of vsv neutralizing antibodies (kelley et al., 1972) . all mice made measurable neutralizing antibody titers to vsv consistent with successful infection. wt vsv administered i.n. produced the highest vsv neutralizing titers (mean titer of 1:8533) consistent with previous results (kapadia et al., 2005) . wt vsv given i.m. and vsv-s given either i.m. or i.n. produced mean vsv neutralizing titers between 1:2560 and 1:3760. the vsvδg-s group produced a lower vsv neutralizing titer (mean of 1:1408) consistent with the fact that this virus does not encode a vsv g protein but does carry g protein on the particles generated by complementation with vsv g. these results indicate that all mice had been effectively inoculated. because a humoral response to the sars-cov s protein is sufficient for protection against sars-cov infection, we wanted to determine the sars-cov neutralizing antibody titers in the serum of mice in this study. in a previous study, we had used direct neutralization of sars-cov to determine sars-cov neutralizing titers. in order to circumvent the level of bio-containment required for this assay, we developed and validated an assay using a vsvδg virus expressing egfp and complemented with sars-cov s protein, the target of sars-cov neutralizing antibodies. we first generated a vsv recombinant, vsvδg-egfp1. the genome of this virus (fig. 1b) has four vsv genes, n, p, l and m, and an egfp gene in the first position of the vsv genome to promote maximal egfp expression. next we inserted the gene for a tagged sars-cov s protein with its cytoplasmic tail replaced with an ha epitope tag (sδtail-ha) into a mammalian expression vector, pcaggs (niwa et al., 1991) . the deletion of the tail is required for infection in the context of pseudotyped viruses (fukushi et al., 2005; giroglou et al., 2004; moore et al., 2004) . this plasmid was transfected into bhk-21 cells. when the transfected cells were expressing sδtail-ha protein, they were in-fected with vsvδg-egfp1 complemented with vsv g. the virus was adsorbed for 1 h, and the cells were then washed three times with pbs in order to remove the input particles. the media was replaced and the infection was allowed to continue for 24 h. the resulting pseudotyped virus, vsvδg-egfp1/sδtail-ha, was present in the media collected from these cells. we next determined if the pseudotyped vsvδg-egfp1/sδtail-ha could be used to assay for sars-cov neutralizing antibodies. we incubated the pseudotyped virus with antiserum from mice inoculated with either wt vsv (which have neutralizing antibody directed to vsv g only), vsv-s (which have neutralizing antibody to vsv and sars-cov), or sars-cov (which have antibody to sars-cov only) at a dilution of 1:50 to ascertain which antibodies were capable of neutralizing the pseudotyped virus. we used vsvδg-egfp1 pseudotyped with vsv g as a control to measure neutralizing antibodies that react with vsv g. following a one-hour incubation at 37°c, the virus-serum mixtures were then transferred to a monolayer of vero e6 cells, which express the sars-cov receptor, ace 2 li et al., 2003) . the cells were incubated at 37°c for 18 h and then fixed with 3% paraformaldehyde. we determined infection by observing egfp expression using fluorescence microscopy. infection of vero e6 cells by the vsvδg-egfp1/sδtail-ha pseudotypes was not neutralized by antibodies to vsv, but was neutralized by antibodies to vsv-s (which contains antibodies to vsv and s) or sars-cov (which contains antibodies to s). in contrast vsvδg-egfp1/g was not neutralized by antibody to sars-cov, but was neutralized by antiserum to vsv or vsv-s (fig. 4) . these results show that neutralization of the s-pseudotyped virus was specific for antibody to sars-cov s. we next compared the sensitivity of our neutralization assay with the standard assay using serum standards assayed previously with the direct sars-cov neutralization assay. we used sera from mice fig. 4 . specific neutralization of vsvδg-egfp1/sδtail-ha by anti-s antibody. vsvδg-egfp1 pseudotyped with either sδtail-ha or vsv g proteins were incubated with antiserum from mice immunized with wt vsv, vsv-s or sars-cov as indicated. the pseudotypes were then transferred to vero e6 cells. infection was determined by egfp expression. both fluorescence images and differential interference contrast (dic) images are shown for each field. immunized with either wt vsv, vsv-s or sars-cov from our previous sars vaccine study (kapadia et al., 2005) . sars-cov neutralizing antibody titers of these sera were determined by incubating vsvδg-egfp/sδtail-ha virus with serial dilutions of these sera, and the virusserum mixtures were transferred to a monolayer of vero e6 cells. infection was determined by observing egfp expression by fluorescence microscopy 18 h after infection. the titer was defined as the highest dilution that completely neutralized vsvδg-egfp1/sδtail-ha. there was no detectable neutralizing activity in serum from mice vaccinated with wt vsv. the titers in serum samples from vsv-s-and sars-cov-inoculated mice were determined to be 1:40 and 1:20 respectively in pseudotype assay. these titers of these sera were 1:32 and 1:12 in the direct assay. furthermore these sera were from mice that were able to control sars-cov infection upon challenge. since an antibody response is sufficient for protecting against sars-cov (bisht et al., 2004; kapadia et al., 2005; yang et al., 2004) , a titer neutralizing titer of as low as 1:20 is indicative of protection. we then used this neutralization assay to measure the neutralizing antibody titers in the serum of the mice in our current study (fig. 5) . no sars-cov-neutralizing antibodies were detected in animals that were infected by wt vsv. there was little variability between individual mice within a group at the three time points measured (fig. 5a, b and c) . notably animals made neutralizing antibodies titers that were considerably greater than 1:20, a titer we determined previously to be protective against sars-cov challenge. animals infected by vsv-s i.n. produced the most robust response with a mean titer of 1:906 five weeks post-vaccination (fig. 5d ). this level dropped by to 1:426 by nine weeks post-vaccination. the group immunized with vsvδg-s also made a strong antibody response with average titers approximately 1:200 at all time points tested. this response was about two-fold greater than that seen in the group immunized i.m. with the replication-competent vsv-s. this difference was statistically significant at 13 weeks post-immunization (p = 0.0087, mann-whitney test). a similar trend was previously reported with a single-cycle vsv vector expressing the hiv env protein. it generally generated a better t-cell response to hiv env than the replication-competent vsv vector expressing hiv env when administered i.m., though the difference was not statistically significant . regulatory approval for the use of replication-competent vsvbased vaccine vectors in humans has been slow because of concerns about potential pathogenesis. we therefore have developed singlecycle vsv-based vectors lacking the vsv g gene that can infect cells, but cannot produce infectious particles (schnell et al., 1997) . we report here that such a replication-defective vector expressing the sars-cov s protein is highly effective at generating sars-cov neutralizing antibody in animals when given i.m. and is even better than a replication-competent vsv vector expressing sars-cov s given by the same route. we were concerned that the single-cycle vsvδg-s vaccine vector described here might be able to mediate multiple rounds of infection because some s protein is incorporated into virions. however, we did not detect any infection by non-g-complemented vsvδg-s particles in cells expressing the sars-cov receptor. furthermore, when we inoculated mice with these non-complemented pseudotyped particles, we saw no immune responses to s or to vsv n indicating that no significant infection occurred. others have also reported that fulllength sars-cov s was not able to mediate entry of vsv and found that a deletion in the carboxy-terminal tail was required for s-mediated entry (fukushi et al., 2005) . the tail of s was also inhibitory in mediating entry of retroviruses (giroglou et al., 2004; moore et al., 2004) . it is likely that the s tail sequence negatively regulates the membrane fusion activity of the s protein, and that in sars-cov virions, other proteins function to activate the s protein membrane fusion activity. consistent with these earlier reports, we found that the full-length s protein would not pseudotype vsvδg-egfp to generate infectious virions, while s protein with its cytoplasmic tail deleted and replaced with an ha tag pseudotyped effectively. taken together, all evidence indicates that vsvδg-s is a single-cycle vector. the strength of the immune response to proteins expressed by replication-competent vsv vectors given i.n. correlates positively with their ability to replicate and spread systemically simon et al., 2007) . single-cycle vectors, which do not spread systemically (simon et al., 2007) , are relatively poor vectors when given i.n., yet generate strong immune responses when given i.m. . in the studies reported here we therefore tested the single-cycle vsvδg-s vector only by the intramuscular route. we found that one dose of the vector was able to generate high levels of sars-cov neutralizing antibody titers of about 1:200. these neutralizing titers were at least ten-fold greater than what was required for complete protection against sars-cov replication in mice in our previous study (kapadia et al., 2005) , and two-fold greater than those induced by the replication-competent vsv-s given i.m.. because antibody responses are sufficient for controlling sars-cov infection (kapadia et al., 2005; yang et al., 2004) , these titers are predictive of protection in the mouse model. although the sars-cov neutralizing antibody titers obtained from mice immunized i.n. with replication-competent vsv-s were higher (average ∼1:600) than the titers obtained from animals immunized i.m., we also know that the replication-competent vectors spread systemically after vaccination by this route (simon et al., 2007) . the virus replicates in the lungs, causes a viremia, and spreads to multiple organs. such widespread dissemination of the vector could also raise safety concerns. how can we explain the greater potency of the single-cycle vectors relative to replication-competent vectors given i.m.? first, we have evidence that replication-competent and single-cycle vectors are both effectively single-cycle vectors when given i.m. (ian simon, unpublished results) . second, the single-cycle vector may be more effective because of the greater expression of s protein in the absence of the upstream g protein gene. because of transcriptional attenuation (iverson and rose, 1981) , the removal of the g gene leads to greater transcription and expression of the sars-cov s gene. in order to evaluate this possibility, we quantified the expression of s (treated with pngase f) by vsv-s and vsvδg-s relative to n/p expression in the gel shown in fig. 1d . we found that vsvδg-s expresses approximately 47% more s protein than vsv-s. lastly, it is also possible that expression of g protein from the replication-competent vector competes with the s protein for the antibody response. the results reported here, along with earlier studies showing potent induction of cellular immune responses by single-cycle vectors , indicate that these single-cycle vectors are excellent alternatives to live-attenuated vsv vaccine vectors and that they warrant further development. to construct pvsvδg-sars s, the sars-cov s gene was amplified from pvsv-sars s (kapadia et al., 2005) by pcr using the following primers: 5′-gatcgatcacgcgtaacatgtttattttcttattatttc-3′ and 5′-cgatccccccgggctagcttatgtgtaatgtaatttgacaccc-3′. the pcr product was digested with mlui and nhei (sites underlined) and ligated to the purified 12,704 bp fragment resulting from the digestion of pvsvxn2 (schnell et al., 1996) with the same enzymes. the plasmid pvsvδg-egfp1 expressing egfp from the first position in the genome was generated by digesting pvsv1xn-egfp with hpai and xbai. the ∼ 12-kb vector fragment was purified and ligated to the ∼ 1.4 kb fragment resulting from the digestion of pvsvδg (roberts et al., 1999) with the same enzymes. the resulting plasmid was designated pvsvδg-egfp1. pcaags-sars sδtail-ha was made by pcr amplification of the sars-cov s gene with primers that replaced the region encoding the last 28 residues of the carboxy-terminal tail with a sequence encoding the ha epitope tag. the following primers were used: 5′-gatc-gatcctcgagaacatgtttattttcttaattatttc-3′ and 5′-cgatc-cccccgggctagcttaggcgtaatctgggacgtcgtatgggtacttgagg-caactgcaacaactagtc-3′. the sequence encoding the ha tag is shown in bold. the resulting pcr product was digested with xhoi and nhei (sites underlined) and ligated into pcaags (niwa et al., 1991) also digested with the same enzymes. construction of pcaags-g was previously described (okuma et al., 2001) . viruses were recovered from plasmids pvsvδg-sars s and pvsvδg-egfp1 by previously described methods (schnell et al., 1997) . the recovered viral supernatants were then transferred onto bhk-21 cells that had been transfected (described below) with pcaags-g (okuma et al., 2001) . the supernatants containing vsvδg-s and vsvδg-egfp1 complemented with g were collected after 36 h. the viruses were titered on bhk-g cells (schnell et al., 1997) using a standard plaque assay. to obtain vsvδg-egfp1 pseudotyped with the sδtail-ha protein, we transfected bhk-21 cell with pcaags-sars sδtail-ha (described below). transfected cells were infected with recovered vsvδg-egfp1 complemented with vsv g. one hour after infection, the input virus was removed and the cells were washed 3 times with phosphate buffered saline (pbs). dmem containing 5% fbs was added to the cells. the media containing vsvδg-egfp1 complemented with sδtail-ha was collected after 36 h. the virus was titered on vero e6 cells by assessing the number of cells expressing egfp. vsv-sars s (vsv-s) (kapadia et al., 2005) and wt vsv (lawson et al., 1995) recovery were previously described. non-complemented vsvδg-s was obtained by infecting bhk-21 cells with vsvδg-s at an moi of 5 for 1 h. the cells were then washed 5 times with pbs to remove any input virus. dmem with 5% fbs was added to the cells and incubated overnight. the media was collected and subjected to ultracentrifugation for 1 h at 100,000 ×g in order to concentrate virus. nine micrograms of dna was diluted in 0.6 ml of optimem (invitrogen, carlsbad, ca), and 30 μl of lipofectamine reagent (invitrogen, carlsbad, ca) was also diluted in 0.6 ml of optimem. the dna and lipofectamine mixtures were combined and incubated for 30 min at room temperature. bhk-21 cells (2 × 10 6 cells plated 18 h earlier) were washed with pbs, and 4.8 ml of optimem was added. the dna/lipofectamine was added to the cells and incubated at 37°c for 5 h. then 6 ml of dulbecco's modified eagle's medium (dmem) containing 20% fetal bovine serum (fbs) was added and left overnight at 37°c. the next morning the media was replaced with dmem containing 5% fbs. the transfection was allowed to continue for 48 h after the addition of the dna/lipofectamine mixture. bhk-21 cells were infected with wt vsv, vsv-s or vsvδg-s at a multiplicity of infection (moi) of 20. after 5 h the cells were washed twice with methionine-free dmem and incubated with 100 μci of [ 35 s]-methionine in 1 ml of methionine-free dmem for 30 min at 37°c. the cells were then washed twice with pbs and solubilized with a detergent solution (1% nonidet p-40, 0.4% deoxycholate, 50 mm tris-hcl [ph 8], 62.5 mm edta). lysates were analyzed by sds-page. the protein samples were treated with peptide n-glycosidase (pngase) f (new england biolabs, beverly, ma) according to manufacturer's instructions. for indirect immunofluorescence microscopy, bhk-21 cells plated on glass coverslips were infected with either wt vsv or vsvδg-s. after 6 h the cells were washed twice with pbs and fixed with 3% paraformaldehyde. the cells were then washed twice with pbs containing 10 mm glycine and incubated with serum from a sars-cov infected mouse at a dilution of 1:200. the coverslips were washed twice with pbs-glycine and incubated with alexa fluor 488 goat anti-mouse igg (molecular probes, eugene, or) diluted 1:500. the cells were washed twice with pbs-glycine and mounted on slides. cells were imaged using a biorad μ radiance confocal scanning system on a nikon elipse te300 microscope with a 60× planapochromat objective. fluorescence microscopy to visualize egfp was performed with a nikon microphot fx microscope equipped with a 40× planapochromat objective, epifluorescence, and a spot digital camera. ten-week-old balb/c mice (charles river laboratories) were used in this study. single intranasal inoculations of 5 × 10 5 plaque forming units (pfu) of wt vsv and vsv-s were administered in a volume of 25 μl to animals lightly anesthetized with 20% isoflurane (baxter, deerfield, il) diluted in propylene glycol (v/v). single intramuscular inoculations of 5 × 10 5 pfu of wt vsv, vsv-s and vsvδg-s were administered in a volume of 50 μl in the hind leg muscle. the vsv neutralization titers are defined as the highest dilution of serum that can completely neutralize infectivity of 100 pfu of vsv on bhk-21 cells. this assay was described previously . in order to measure sars-cov neutralizing antibodies in serum, vsvδg-egfp1/ sδtail-ha was first incubated with two monoclonal antibodies, i1 and i14 (lefrancois and lyles, 1982) , at a dilution of 1:1000 per antibody for 1 h at 37°c to neutralize potential infection due to any residual vsv g that may have been incorporated into the particles pseudotyped with sδtail-ha protein. serum samples were serially diluted with dmem containing 5% fbs. approximately 75 infectious pseudotyped particles were added to each serum dilution in a final volume of 30 μl. the mixture was incubated for an hour at 37°c. 25 μl of each dilution was transferred to a monolayer of vero e6 cells grown in a 96-well plate. after 1 h at 37°c, 50 μl of dmem with 5% fbs was added to each well and the cells were incubated for 16 to 20 h at 37°c. infection was determined by visualizing egfp expression using an olympus ck40 microscope equipped for epifluorescence. each dilution was measured in duplicate. the titer was determined to be the highest dilution at which both duplicates showed no infection. severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment identification of a novel coronavirus in patients with severe acute respiratory syndrome immunogenicity of attenuated vesicular stomatitis virus vectors expressing hiv type 1 env and siv gag proteins: comparison of intranasal and intramuscular vaccination routes vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein development of a new vaccine for the prevention of lassa fever emerging respiratory viruses: challenges and vaccine strategies retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein isolation and characterization of viruses related to the sars coronavirus from animals in southern china localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (rsv) antigens protect mice against rsv challenge longterm protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine the glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats recombinant vesicular stomatitis viruses from dna the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus ii. monoclonal antibodies of nonneutralizing and cross-reactive epitopes of indiana and new jersey serotypes angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses the genome sequence of the sars-associated coronavirus retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2 recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type 2 gd elicit robust cd4+th1 immune responses and are protective in mouse and guinea pig models of vaginal challenge efficient selection for high-expression transfectants with a novel eukaryotic vector analysis of the molecules involved in human t-cell leukaemia virus type 1 entry by a vesicular stomatitis virus pseudotype bearing its envelope glycoproteins an optimized vaccine vector based on recombinant vesicular stomatitis virus gives high-level, long-term protection against yersinia pestis challenge severe acute respiratory syndrome a single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector highly effective control of an aids virus challenge in macaques by using vesicular stomatitis virus and modified vaccinia virus ankara vaccine vectors in a single-boost protocol a vesicular stomatitis virus recombinant expressing granulocyte-macrophage colony-stimulating factor induces enhanced t-cell responses and is highly attenuated for replication in animals epizootic vesicular stomatitis in colorado, 1982: infection in occupational risk groups intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus l1 protein provides complete protection against papillomavirus-induced disease vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge attenuated vesicular stomatitis viruses as vaccine vectors complete protection from papillomavirus challenge after a single vaccination with a vesicular stomatitis virus vector expressing high levels of l1 protein an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants characterization of a novel coronavirus associated with severe acute respiratory syndrome successful vaccine-induced seroconversion by single-dose immunization in the presence of measles virus-specific maternal antibodies successful mucosal immunization of cotton rats in the presence of measles virusspecific antibodies depends on degree of attenuation of vaccine vector and virus dose the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus construction of a novel virus that targets hiv-1-infected cells and controls hiv-1 infection replication and propagation of attenuated vesicular stomatitis virus vectors in vivo: vector spread correlates with induction of immune responses and persistence of genomic rna a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this work was supported by nih grant ai057158. key: cord-270380-1me7ugkg authors: wang, xiaona; li, fengsai; han, meijing; jia, shuo; wang, li; qiao, xinyuan; jiang, yanping; cui, wen; tang, lijie; li, yijing; xu, yi-gang title: cloning, prokaryotic soluble expression, and analysis of antiviral activity of two novel feline ifn-ω proteins date: 2020-03-19 journal: viruses doi: 10.3390/v12030335 sha: doc_id: 270380 cord_uid: 1me7ugkg cats are becoming more popular as household companions and pets, forming close relationships with humans. although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. interferons (ifns), especially type i ifns (ifn-α, ifn-β, and interferon omega (ifn-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. nevertheless, there is limited knowledge regarding feline ifn-ω (feifn-ω), compared to ifn-α and ifn-β. in this study, we cloned the genes encoding feifn-ωa and feifn-ωb from cat spleen lymphocytes. homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feifn-ω. the recombinant feifn-ωa and feifn-ωb proteins were expressed in their soluble forms in escherichia coli, followed by purification. both proteins exhibited effective anti-vesicular stomatitis virus (vsv) activity in vero, f81 (feline kidney cell), madin–darby bovine kidney (mdbk), madin–darby canine kidney (mdck), and porcine kidney (pk-15) cells, showing broader cross-species antiviral activity than the intercat ifn antiviral drug. furthermore, the recombinant feifn-ωa and feifn-ωb proteins demonstrated antiviral activity against vsv, feline coronavirus (fcov), canine parvovirus (cpv), bovine viral diarrhea virus (bvdv), and porcine epidemic diarrhea virus (pedv), indicating better broad-spectrum antiviral activity than the intercat ifn. the two novel feifn-ω proteins (feifn-ωa and feifn-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats. as cats continue to become more popular as household companions and pets [1] , a variety of viral infections pose a serious threat to felines, including a high incidence of feline leukemia virus (felv) [2] , feline coronavirus (fcov) [3] , feline immunodeficiency virus (fiv) [4] , and feline panleukopenia virus (fpv) [5] . currently, the only preventative vaccines and therapeutic drugs available for use in pet cats have limited effectiveness. nevertheless, interferons (ifns) play an increasingly complementary role in homology and phylogenetic tree analysis of the feline ifn-ω genes isolated in this study were analyzed using dnastar and mega7 software. in addition, the characteristics of the feifn-ω genes and proteins were analyzed by several online bioinformatics software programs: signal peptide cleavage sites were analyzed by the signalp 3.0 server at http://www.cbs.dtu.dk/services/signalp-3.0/; phosphorylation sites were analyzed by the netphos3.1 server at http://www.cbs.dtu.dk/services/netphos/; n-glycosylation sites were analyzed by the netnglyc1.0 server at http://www.cbs.dtu.dk/services/netnglyc/; o-glycosylation sites were analyzed by the yinoyang1.2 server at http://www.cbs.dtu.dk/services/yinoyang/; subcellular localization was analyzed by the targetp 1.1 server at http://www.cbs.dtu.dk/services/targetp; transmembrane regions were analyzed by the tmhmm 2.0 server at http://www.cbs.dtu.dk/services/tmhmm/; antigen epitopes and hydrophobicity were analyzed by the bepipred 1.0 server at http://www.cbs.dtu. dk/services/bepipred-1.0/; and secondary and three-dimensional structures were predicted by sopma at https://npsa-prabi.ibcp.fr/cgi-bin/npsa. in this study, the genes encoding feifn-ω that were isolated by rt-pcr were subcloned as a kpni and bamhi-generated (new england biolabs, ma, usa) gene fragment into the prokaryotic soluble expression plasmid pcold-tf, giving rise to recombinant pcold-feifn-ω. after that, the recombinant plasmid was transformed into e. coli bl21 (de3) competent cells and validated by pcr and sequencing analyses, thus generating the recombinant e. coli strain pcold-feifn-ω/bl21. we then used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) analysis to determine the optimal conditions for expression of the recombinant feifn-ω in pcold-feifn-ω/bl21 cells via induction by isopropyl β-d-thiogalactoside (iptg, sigma, st. louis, mo, usa). briefly, for optimization of the iptg concentration, the recombinant e. coli strain was grown in luria-bertani broth (sigma, st. louis, mo, usa) supplemented with 100 µg/ml ampicillin at 37 • c until the optical density at 600 nm was approximately 0.5. then, iptg was added at final concentrations of 0.4 mmol/l, 0.6 mmol/l, 0.8 mmol/l, 1.0 mmol/l, or 1.2 mmol/l, and the cultures were continually cultivated for viruses 2020, 12, 335 4 of 16 another 6 h. after centrifugation at 12,000× g for 10 min, the cell pellets were lysed and analyzed by 12% sds-page. to determine the optimal induction time, the recombinant strain was induced by the optimized final concentration of iptg for 4 h, 6 h, 8 h, 10 h, and 12 h. following centrifugation and cells lysis, the proteins were again analyzed by 12% sds-page. the fusion protein expressed by the pcold-feifn-ω/bl21 bacteria cells was subjected to cleavage by the 3c protease (sigma, st. louis, mo, usa). the target recombinant feifn-ω protein was then purified using ni 2+ affinity chromatography columns according to the manufacturer's instructions, followed by confirmation analysis using sds-page. the purified feifn-ω protein was stored at −80 • c until use. vsv was used as a virus model to evaluate the antiviral activity of the recombinant feifn-ω via the in vitro microdose cytopathic effect inhibition assay (mcia) according to the method described previously [12, 26] with slight modifications. briefly, 100 µl of the purified feifn-ω sample was serially diluted ten-fold in dmem containing 10% fbs and transferred to confluent f81 cell monolayers in 96-well cell culture plates, and then incubated at 37 • c in 5% co 2 for 18 h. f81 cells without ifn treatment were used as a control group. after incubation, vsv (moi = 1) was prepared as described above, added into the 96-well plate, and incubated for 8-12 h until the cpe of the cells in the viral control group reached 100%. next, the culture medium was removed, and the cells were stained with 0.2% crystal violet in 20% ethanol at 37 • c for 30 min. the cells were then destained with 0.1% acetic acid in 50% ethanol at 37 • c for 5 min before determining the absorbance of each well at 595 nm. each sample was performed with eight biological replicates and three technical replicates. the antiviral activity of the ifn was calculated as the method previously described, which is expressed as units per milligram according to the ratio of ifn titer and protein concentration [27] . in parallel, the intercat ifn antiviral drug (toray industries, tokyo, japan) was used as an ifn treatment control. in addition, we determined the species-specific antiviral activity of the recombinant feifn-ω by conducting mcias using vsv in f81, vero, mdck, mdbk, and pk-15 cells. broad-spectrum antiviral activity of the recombinant feifn-ω was determined by conducting mcias using vsv, fcov, cpv, bvdv, and pedv. the results are shown as mean ± sem (n = 3) for three independent experiments. statistical analyses were performed using graphpad prism v5.0 software. tukey's multiple comparison tests and one-way analysis of variance (anova) tests were used to analyze the significance of the differences between groups: * p < 0.05; ** p < 0.01; and *** p < 0.001. we first collected splenic lymphocytes from cats infected with vsv and treated with poly(i:c) simultaneously. we then performed rt-pcr assays using a pair of degenerate primers to identify two genes encoding feifn-ω, referred to as feifn-ωa and feifn-ωb ( figure 1a) . these two novel genes were deposited in the genbank with accession numbers mk682680 and mk682681. following nucleotide sequence homology analysis, we found that the feifn-ωa gene (mk682680), with a size of 591 bp, shared a maximum nucleotide sequence homology of 91.88% (91.6% amino acid homology) with the 13 known subtypes of feifn-ω (feifn-ω1 to feifn-ω13) genes published in the genbank (figure 1c ). the feifn-ωb gene (mk682681), with a size of 612 bp, shared a maximum sequence homology of 90.20% (89.4% amino acid homology) with the 13 known subtypes of feifn-ω ( figure 1d ). the sequence homology shared between the feifn-ωa and feifn-ωb genes was only 83.01% (figure 1b) , indicating that the two genes identified in this study were novel feifn-ω genes. we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega7. the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure 2 ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. viruses 2020, 12, x for peer review 6 of 16 we constructed a phylogenetic tree comparing the feifn-ωa/ωb genes identified in this study with other ifn gene sequences published in genbank from different animals using the software dnastar (megalign) and mega7. the results showed that the feifn-ωa/ωb genes belonged in the type i ifn family, but that their evolutionary relationship to the known feline ifn-ω genes already published in the genbank (figure 2 ) was distant, indicating that the feifn-ωa and feifn-ωb genes, identified for the first time here, are new subtypes of feline ifn-ω. the characteristics of the novel feline ifn-ω proteins (feifn-ωa and feifn-ωb) were analyzed using several online bioinformatics software programs, including the identification of potential signal peptide cleavage sites, n-glycosylation sites, o-glycosylation sites, phosphorylation sites, subcellular localization, and transmembrane regions. the results from these detailed analyses are displayed in table 1 . in addition, we also used online software algorithms to predict antigen epitopes, hydrophobicity, and the secondary and three-dimensional structures of the feifn-ωa and feifn-ωb proteins. as shown in figure figure 3b ). the maximum hydrophobicity of feifn-ωa was 1.92, and the minimum hydrophobicity was −2.27. the maximum hydrophobicity of feifn-ωb was 2.27, and the minimum hydrophobicity was −2.36. the secondary structure of the two feifn-ω proteins was predicted using sopma software and revealed that feifn-ωa contained 62.24% alpha helix, 2.55% beta sheet, and 34.18% irregular curl structures (figure 3c ), whereas feifn-ωb contained 65.52% alpha helix, 1.97% beta sheet, and 31.53% irregular curl structures ( figure 3d ). the three-dimensional structures of feifn-ωa and feifn-ωb were predicted with swiss-model software and are shown in figure 3e ,f, respectively. (6) 84.3% extracellular 9.2% intracellular 7% mitochondrion intracellular a signal peptide cleavage sites were analyzed by signalp 3.0 server at http://www.cbs.dtu.dk/services/ signalp-3.0/; b phosphorylation sites were analyzed by netphos3.1 server at http://www.cbs.dtu.dk/services/ netphos/; c n-glycosylation sites were analyzed by netnglyc1.0 at http://www.cbs.dtu.dk/services/netnglyc/ and o-glycosylation sites were analyzed by yino yang1.2 at http://www.cbs.dtu.dk/services/yinoyang/; d subcellular localization was analyzed by targetp 1.1 server at http://www.cbs.dtu.dk/services/targetp; e transmembrane region was analyzed by tmhmm server v. 2.0 at http://www.cbs.dtu.dk/services/tmhmm/. the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl21 competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl21 and pcold-feifn-ωb/bl21. after inducing their expression with iptg, the rfeifn-ωa ( figure 4a ) and rfeifn-ωb (figure 4b ) proteins fused with the trigger factor (tf) of approximately 57 kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl21 and pcold-feifn-ωb/bl21 e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of 1.0 mmol/l ( figure 4c ) and an induction time of 8 h (figure 4d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of 0.8 mmol/l ( figure 4e ) and an induction time of 10 h (figure 4f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni 2+ affinity column chromatography and subjected to cleavage using the 3c protease. the cleaved proteins were then purified by his-tag ni 2+ affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately 21 kda and 22 kda, respectively ( figure 4g ). viruses 2020, 12, x for peer review 9 of 16 the genes encoding feifn-ωa and feifn-ωb were subcloned into the prokaryotic soluble expression vector pcold-tf and then transformed into e. coli bl21 competent cells, thus generating the recombinant protein expressing e. coli strains pcold-feifn-ωa/bl21 and pcold-feifn-ωb/bl21. after inducing their expression with iptg, the rfeifn-ωa ( figure 4a ) and rfeifn-ωb (figure 4b ) proteins fused with the trigger factor (tf) of approximately 57 kda and were primarily expressed in their soluble forms. subsequently, we optimized the expression conditions of the rfeifn-ω proteins from the pcold-feifn-ωa/bl21 and pcold-feifn-ωb/bl21 e. coli strains. our observations indicated that the optimal induction conditions for rfeifn-ωa protein expression were an iptg concentration of 1.0 mmol/l (figure 4c ) and an induction time of 8 h (figure 4d) , and the optimal induction conditions for rfeifn-ωb protein expression were an iptg concentration of 0.8 mmol/l (figure 4e ) and an induction time of 10 h (figure 4f ). after that, the fused proteins (rfeifn-ωa/ωb+tf) were purified by his-tag ni 2+ affinity column chromatography and subjected to cleavage using the 3c protease. the cleaved proteins were then purified by his-tag ni 2+ affinity column chromatography once again, after which we collected the purified recombinant rfeifn-ωa and rfeifn-ωb proteins with molecular weights of approximately 21 kda and 22 kda, respectively (figure 4g ). we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f81 cells. as shown in figure 5 , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. viruses 2020, 12, x for peer review 10 of 16 the black arrow represents the target fusion protein and red arrowhead represents the target fusion protein expressed under the optimized condition. we determined the antiviral activity of rfeifn-ωa and rfeifn-ωb using microdose cytopathic effect inhibition assays (mcias) and vsv as the viral model that was propagated on f81 cells. as shown in figure 5 , both rfeifn-ωa and rfeifn-ωb demonstrated good antiviral activity similar to the intercat ifn (feline ifn antiviral drug) positive control. however, no antiviral effect in negative control groups (cells untreated with the ifns) was detected. we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f81 cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk-15 cells (pig). as shown in figure 6 , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f81 cells) and heterologous animal cells (vero, mdck, mdbk, and pk-15 cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f81 and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk-15 cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). we then tested whether the antiviral activities of rfeifn-ωa and rfeifn-ωb were species-specific. to address this question, we performed mcias with vsv propagated in several different cell lines from different animal species, including f81 cells (cat), vero cells (monkey), mdck cells (dog), mdbk cells (cattle), and pk-15 cells (pig). as shown in figure 6 , the purified recombinant feifn-ωa and feifn-ωb proteins showed antiviral activity in both homologous animal cells (f81 cells) and heterologous animal cells (vero, mdck, mdbk, and pk-15 cells) in vitro. the antiviral activity of the rfeifn-ω proteins in heterologous animal cells was significantly stronger than that of the intercat ifn control. although the intercat ifn displayed high antiviral activity in f81 and vero cells, it had significantly weak antiviral activity in mdck and mdbk cells, and no antiviral activity in pk-15 cells. furthermore, the antiviral activity of rfeifn-ωb was better than that of rfeifn-ωa in homologous and heterologous animal cells, but especially in homologous animal cells that originated from cats. however, no antiviral activity was detected in cells' control groups (cells untreated with the ifns). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure 7 , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure 7 . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure 6 . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < 0.05; *** p < 0.001) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < 0.05). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure 7 , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). viruses 2020, 12, x for peer review 11 of 16 figure 6 . the species-specific antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < 0.05; *** p < 0.001) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < 0.05). next, we wanted to investigate whether the antiviral activities of the recombinant feifn-ωa and feifn-ωb proteins were also broad-spectrum. we performed mcias using a wide range of different viruses, including vsv, fcov, cpv, bvdv, and pedv. as shown in figure 7 , rfeifn-ωa and rfeifn-ωb both displayed in vitro antiviral activity against vsv, fcov, cpv, bvdv, and pedv. their antiviral activities were especially strong against fcov and vsv. in contrast, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of rfeifn-ωa. no notable differences were observed between rfeifn-ωa and rfeifn-ωb with cpv, bvdv, or pedv. in addition, the antiviral activity of rfeifn-ωb against fcov and vsv was significantly stronger than that of intercat ifn, and the antiviral activities of both rfeifn-ωa and rfeifn-ωb against cpv, bvdv, and pedv were significantly higher than that of intercat ifn. in fact, we observed no antiviral activity against bvdv or pedv by intercat ifn. unsurprisingly, there was no antiviral activity detected in cells' control groups (cells untreated with the ifns). figure 7 . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the figure 7 . the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb using intercat ifn for comparison. bars represent the mean ± sem for each group. significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) were observed when comparing the rfeifn-ωa/rfeifn-ωb groups to the intercat ifn group. a comparison of the rfeifn-ωb group and rfeifn-ωa group also indicated significant differences (# p < 0.05). currently, cats are continuing to become more and more common as household pets. we know of a wide variety of viral infections that can seriously endanger the health of pet cats, such as felv, fiv, fipv, fcov, and feline calicivirus (fcv). interferon represents a promising potential therapeutic agent that can effectively treat pet cats infected with these viruses. in this study, we identified two new genes encoding feline ifn-ω (feifn-ωa and feifn-ωb) in the spleen lymphocytes of cats. following sequence homology analysis, we found that feifn-ωa and feifn-ωb shared maximum nucleotide sequence homologies of 91.88% and 90.20%, and maximum amino acid homologies of 91.6% and 89.4% with the 13 previously published subtypes of feline ifn-ω, respectively. in addition, phylogenetic tree analysis of ifns in cats and other species constructed using neighbor-joining analysis revealed that feifn-ωa and feifn-ωb do belong to the type i ifn family, but their evolutionary relationship to known feline ifn-ω genes was distant, indicating that feifn-ωa and feifn-ωb were indeed new subtypes of feline ifn-ω. we deposited these two genes into the genbank with the accession numbers mk682680 (feifn-ωa) and mk682681 (feifn-ωb), thus enriching the ifn-ω data submitted to the genbank [12] . in this study, the characteristics of the feifn-ωa and feifn-ωb proteins, such as signal peptide sequences, signal peptide cleavage sites, phosphorylation sites, glycosylation sites, antigen epitopes, hydrophobicity, and transmembrane regions were analyzed using bioinformatics to provide better theoretical guidance for the functional study of these proteins. the mature proteins, with normal biological activity, were formed only after the signal peptide sequence was removed from the precursor protein, thus allowing them to be secreted outside the cell membrane [28, 29] . we used online software to predict that the signal peptide sequence of the feifn-ωa/ωb proteins consists of 23 amino acid residues, and that the signal peptide cleavage site is located between residues gly23 and cys24. the results indicated that the recombinant feifn-ωa and feifn-ωb proteins could be expressed in vitro in their soluble forms. glycosylation is an important post-translational modification process that can affect the antigenic determinants, charge properties, enzymatic properties, and thermal stability of proteins. similar to previous reports for the 13 known feifn-ω subtypes [12] , we observed no n-glycosylation sites present in feifn-ωa or feifn-ωb. however, our analyses predicted nine potential o-glycosylation sites in feifn-ωa and six potential o-glycosylation sites in feifn-ωb, which is different from previous reports on the other known ifn-ω subtypes [6] . studies have shown that glycosylation sites can play an important role in determining the activity of ifns [30, 31] . for example, glycosylated ifn-ω has been observed to be markedly more potent than non-glycosylated ifn-ω against hepatitis c virus, bvdv, yellow fever virus, and west nile virus, with even more superior effects than ifn-α, ifn-β, and ifn-γ [6, 30] . insect/baculovirus expression systems are one of the most effective eukaryotic expression systems for preparing feline ifns. these expression systems are capable of making post-translational modifications, such as glycosylation, which allows proteins to fold correctly, thus producing highly active and stable ifns [32] [33] [34] [35] [36] . however, the application of these systems is limited by drawbacks, such as low yield of ifn protein and complicated operation requirements [37] . therefore, e. coli expression systems are still the most widely used prokaryotic expression system for protein production with their characteristics of simple procedures, large-scale production, and low cost [38] [39] [40] . generally, recombinant ifn is produced by e. coli expression systems in an insoluble inclusion body form that has not been modified or folded correctly, resulting in the loss of its biological activity [41, 42] . to obtain a soluble and biologically active ifn protein, the inclusion bodies need to be denatured and then renatured, which is a time-consuming, laborious process [40] . in this study, we selected the prokaryotic soluble expression system pcold-tf to prepare recombinant feifn-ωa and feifn-ωb. the pcold-tf system is a highly efficient soluble expression system with a his-tagged tf that allows the expressed protein to be effectively modified, folded, and secreted into the cytoplasm [43, 44] . following the construction of recombinant e. coli strains and induction by iptg, the rfeifn-ωa/ωb proteins were expressed in their soluble forms and analyzed via sds-page. we optimized expression conditions for the rfeifn-ωa and rfeifn-ωb proteins and purified them using his-tag ni 2+ affinity column chromatography. both proteins showed antiviral activity against vsv in microdose cytopathic effect inhibition assays using f81 cells. our results provide a basis for further studies into the development of rfeifn-ωa and rfeifn-ωb as therapeutic agents for viral infections. meanwhile, our data provide evidence that the pcold-tf expression system can be used to produce ifn with full bioactivity. recombinant feline interferon-ω (rfeifn-ω) was the first licensed immunomodulator for use in treating viral infections in cats, especially fiv and felv infections [17, 18, 45] . furthermore, rfeifn-ω also exhibits therapeutic effects against other feline viral infections, such as fcv, feline parvovirus, and feline herpesvirus-1 [45] , as well as viruses that originate in other animals, such as foot-and-mouth disease virus, influenza virus, bvdv, vsv, prv, and rotavirus [6, 12, 18] . in this study, our results demonstrated that both rfeifn-ωa and rfeifn-ωb had antiviral activity in homologous animal cells (f81 cells, cat) and heterologous animal cells (vero cells, monkey; mdbk cells, cattle; mdck cells, dog; pk-15 cells, pig), indicating that rfeifn-ωa and rfeifn-ωb have broad cross-species antiviral activity in vitro, similar to previously published findings in mdbk and mdck cells [12] . in contrast, the antiviral activities of rfeifn-ωb in homologous and heterologous animal cells were better than that of rfeifn-ωa. intriguingly, the rfeifn-ωa and rfeifn-ωb proteins exhibited antiviral activity in mdck cells, despite the absence of ifn-ω in canines [46, 47] . we speculate that different ifns with different physiological functions are likely responsible for the difference of results obtained in our study compared to other published studies. furthermore, analysis of the broad-spectrum antiviral activities of rfeifn-ωa and rfeifn-ωb revealed that they were effective against vsv, fcov, pedv, bvdv, and cpv. out of those five viruses, antiviral activity was strongest against vsv and fcov. no significant differences in antiviral activity against cpv, bvdv, or pedv were observed between rfeifn-ωa and rfeifn-ωb. however, the overall broad-spectrum antiviral activity of rfeifn-ωb was significantly stronger than that of rfeifn-ωa. recently, combination antiviral therapy has become a common practice in treating feline viral infections due to pharmacokinetics and the short half-life of ifn alone [48, 49] . there are many published studies focused on the combination of ifn-ω with other therapeutic agents, such as chemotherapeutic agents [50] , ifn-α [51] , and ribavirin [6] , suggesting this is an attractive strategy to use against viral infections. we further evaluated the antiviral effects of rfeifn-ωa and rfeifn-ωb combined with feline il-18 against vsv and fcov in f81 cells. our results revealed that the in vitro antiviral activity of combination therapy was significantly increased compared to that of rfeifn-ωa or rfeifn-ωb alone, indicating that rfeifn-ω combination therapy may represent a more potent option than monotherapy for treating viral infections in cats; however, this hypothesis requires further exploration in vivo. in summary, the rfeifn-ωa and rfeifn-ωb obtained in this study exhibit significant broad-spectrum antiviral activity in both homologous and heterologous cells in vitro, particularly rfeifn-ωb, suggesting a promising candidate for the development of an effective therapeutic agent against viral infections in cats and other animals. in this study, two new subtypes of feline ifn-ω (ωa and ωb) were identified and characterized. they shared a maximum nucleotide sequence homology of 91.88% and 90.20% and a maximum amino acid homology of 91.6% and 89.4% with the 13 previously known subtypes of feifn-ω, respectively. we analyzed the characteristics of feifn-ωa and feifn-ωb in detail using bioinformatics followed by soluble expression and optimization of induction conditions in e. coli. our data showed that purified recombinant feifn-ωa and feifn-ωb had broad-spectrum antiviral activity in homologous and heterologous animal cells, suggesting they are candidates for the development of effective therapeutic agents to be used against viral infections in pet cats. and, our research is underway to systematically evaluate the effectiveness of the two novel feifns as therapeutics agent for cat viral infections in vivo. in addition, the reported feifn-ω sequences will enrich the ifn data submitted to genbank. epidemiology of naturally occurring feline urologic syndrome feline leukemia virus infection: importance and current situation in switzerland effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis the protective rate of the 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dna encoding human thymosin α1and interferon ω1 potently inhibits liver tumor growth in icr mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-324674-yd7idp90 authors: zhang, chengfei; yan, yan; he, hongwang; wang, li; zhang, na; zhang, jie; huang, hongjun; wu, nannan; ren, hua; qian, min; liu, mingyao; du, bing title: ifn-stimulated p2y(13) protects mice from viral infection by suppressing the camp/epac1 signaling pathway date: 2018-08-22 journal: j mol cell biol doi: 10.1093/jmcb/mjy045 sha: doc_id: 324674 cord_uid: yd7idp90 among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. however, the function of p2 receptors in viral infection has been little explored. here we demonstrated that p2y(13) and its ligand adp play an important role in protecting hosts from viral infections. first, we demonstrate that p2y(13), as a typical interferon-stimulated gene, is induced together with extracellular adp during viral infection. most importantly, extracellular adp restricts the replication of different kinds of viruses, including vesicular stomatitis virus, newcastle disease virus, herpes simplex virus 1, and murine leukemia virus. this kind of protection is dependent on p2y(13) but not p2y(1) or p2y(12), which are also considered as receptors for adp. furthermore, cyclic adenosine monophosphate and epac1 are downregulated by extracellular adp through the p2y(13)-coupled gi alpha subunit. accordingly, inhibition or deletion of epac1 significantly eliminates adp/p2y(13)-mediated antiviral activities. taken together, our results show that p2y(13) and adp play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets. upon viral infection, the host can sense the virus through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, especially toll-like receptors (tlrs) and rig-i-like receptors (rlrs) (kawai and akira, 2010; chen et al., 2013) . when activated by viral nucleic acids, tlrs and rlrs will activate the tbk1-irf3 axis, leading to type i interferon (ifn) expression and secretion (garcia-sastre and biron, 2006; he et al., 2017) . then, the released ifns induce hundreds of interferon-stimulated genes (isgs) via the janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway to clear the invaded virus (platanias, 2005; garcia-sastre and biron, 2006; shu et al., 2015) . among them, the double-stranded rna-dependent protein kinase pkr, the myxovirus resistance gene mx, the 2′-5′ oligoadenylate synthetases, and interferon induced transmembrane protein 3 (ifitm3) have been shown to disrupt viral infection in various manners rafique et al., 2011; liu et al., 2013) . more than 350 genes have been characterized as isgs. however, their roles and mechanisms in fighting against multiple distinct classes of viruses remain largely unexplored. purinergic signaling and purinergic receptors are important in both innate and adaptive immune responses. purinergic signaling was first reported in 1929 and, after several decades of study, many of the biologic effects of atp, utp, adp, udp, and adenosine signaling were discovered (eltzschig et al., 2012; idzko et al., 2014) . in tissue injury or pathogen infection, nucleotides can be released from stressed cells as a danger signal, alerting the cells by interaction with purinergic receptors in an autocrine or paracrine manner . nineteen different purinergic receptor subtypes have been identified, which can be classified into p1 and p2 receptors. four p1 receptor subtypes can be activated by adenosine. p2 receptors can be further divided into p2y receptors (p2yrs) and p2x receptors (p2xrs) based on their structures and distinct signaltransduction mechanisms. p2yrs are g-protein-coupled receptors for atp, adp, utp, and udp, while p2xrs are nucleotide-gated ion channels and can be activated by atp (ralevic and burnstock, 1998; junger, 2011; idzko et al., 2014) . many studies revealed crucial roles for p2 receptors during inflammatory and infectious diseases. for example, atp-triggered p2x7 activation is crucial for the activation of nlrp3 inflammasome and the subsequent release of interleukin-1β (di virgilio et al., 2017) . atp/utp receptor p2y 2 signaling has been identified as a 'find-me' signal to recruit motile phagocytes, thereby promoting clearance of apoptotic cells or bacteria (elliott et al., 2009; chen et al., 2010) . our previous study demonstrated that the adp-mediated p2y 12 /p2y 13 signal pathway protects the host defense against bacterial infection via erk signaling (zhang et al., 2018) . however, whether adp-(or its receptors-) mediated immune regulation is also involved in viral infection remains unclear. p2y 1 , p2y 12 , and p2y 13 are all receptors for adp. p2y 1 binds to gtp-binding protein subunit alpha q (gq), which can evoke calcium signaling. while p2y 12 and p2y 13 interact with gtpbinding protein subunit alpha i (gi), which can suppress the activity of adenylate cyclase (adcy) and the production of cyclic adenosine monophosphate (camp) (zhang et al., 2015; shinozaki et al., 2017) . in this study, we demonstrate that p2y 13 , as an isg, and its ligand, adp, is released from infected cells as a danger signal during viral infection, which restricts the replication of both dna and rna viruses. adp/p2y 13 -mediated protection against viral infection operates by suppressing the expression of exchange protein activated by camp 1 (epac1), which is an alternative key intracellular sensor for camp. therefore, we demonstrated that adp/p2y 13 restricts viral infection through suppressing the camp/epac1 signaling pathway, which has potential therapeutic significance in the prevention and control of viral diseases. the expression of p2y 13 is increased by ifn through jak/stat pathway to identify p2y family members potentially involved in antiviral immune responses, we treated mouse bone marrowderived macrophages (bmdms) with ifn-α, ifn-β, and ifn-γ to appraise the rna expression of all p2y family members by quantitative real-time pcr (q-pcr). as shown in figure 1a , the rna expression of p2y 13 was upregulated obviously by all three ifns. the protein level of p2y 13 also upregulated by ifn-β ( figure 1b) . besides, when blocked ifn receptor (ifnar) with the anti-ifnar antibody, the upregulated expression of p2y 13 was decreased ( figure 1c ), suggesting that p2y 13 upregulation is dependent on signals downstream of ifn. the jak/stat pathway is crucial for ifn-induced cellular antiviral signaling. to determine whether ifn-induced p2y 13 upregulation via the jak/stat1 pathway, we pretreated mouse peritoneal macrophages (pems) with ruxolitinib (a jak1 inhibitor) and fludarabine (a stat1 inhibitor) and found that ifnβ-induced p2y 13 expression was blocked by both inhibitors ( figure 1d and e). inhibition of stat1 also reduced vsv-induced p2y 13 expression ( figure 1f ). in addition, knockout of tlr3 to block tlr3-mediated activation of ifn also suppressed poly(i:c)-induced p2y 13 expression ( figure 1g ). taken together, these data demonstrated that p2y 13 has the typical characteristics of isgs. as an endogenous ligand to purinergic receptors, adp can be released from injured cells to activate p2y 1 , p2y 12 , and p2y 13 receptors. to investigate the potential role of adp as a danger signal in viral infection, the release of endogenous adp in viralinfected cells was measured. as shown in figure 2a and b, extracellular adp in vsv-infected cell supernatant was clearly increased in a time-dependent manner. besides, ndv-infected bmdms and hsv-1-infected hek293t cells also release large amounts of adp ( figure 2c and supplementary figure s1a ), suggesting adp can be released as a danger signal during viral infection. to investigate the biological significance of adp, we pretreated the cells with adp before vsv infection. to our surprise, the rna replication of vsv in adp-treated raw264.7 cells was reduced significantly in a time-( figure 2d ) and concentration-( figure 2e ) dependent manner. accordingly, the viability of vsv-infected raw 264.7 cells was increased dramatically by adp in a concentration-dependent manner while have little influence on uninfected cells ( figure 2f and supplementary figure s1b ), indicating that extracellular adp protects cells from vsv infection. to confirm whether adp has broad-spectrum antiviral activities, raw264.7 cells and bmdms were pretreated with adp before vsv, ndv, and hsv-1 infection. as shown in figure 2g -j and supplementary figure s1c , the rna expression of vsv, ndv, hsv-1 and the titers of vsv were all strikingly decreased by adp. similar results were observed in hek293t cells, where cell cytopathic effects were induced by vsv and hsv-1, as determined using a crystal violet staining assay (supplementary figure s1d and e). we further infected hek293t and hela cells with vsv-gfp and mlv-gfp and found that the number of gfppositive cells was decreased in adp-treated cells, indicating that vsv-gfp and mlv-gfp were restricted by adp (supplementary figure s1f-i). to rule out the possibility that adp degradation products mediate the suppression of viral replication, we treated cells with non-hydrolyzable adp analog adp-β-s. as shown in supplementary figure s1j , adp-β-s has a similar role as adp in restricting vsv replication. taken together, our data suggested that extracellular adp has broad-spectrum antiviral activities, which have great potential in protecting hosts from a viral infection. to explore the key receptors involved in adp-mediated antiviral activities, we detected the expression of p2y 1 , p2y 12 , and p2y 13 after vsv infection. as shown in figure 3a , only the expression of p2y 13 was increased significantly, whereas the expression of p2y 1 and p2y 12 was little changed in vsv-infected raw264.7 cells. similar data were observed in poly(i:c)-treated bmdms ( figure 3b ). we then used mrs 2179 and mrs 2211 to inhibit p2y 1 and p2y13, respectively, to verify whether p2y 1 and p2y 13 contributed to adp-mediated antiviral activities. we found that adp-mediated antiviral activities were little influenced by mrs 2179 but significantly decreased by mrs 2211 as shown in figure 3c -f, and both of mrs 2179 and mrs 2211 have little influence on cell viability (supplementary figure s2a and b). consistently, when infected wild-type, p2y 1 -, p2y 12 -, and p2y 13deficient bmdms with vsv, the vsv rna replication was increased only in p2y 13 -deficient bmdms, not in p2y 1 -or p2y 12 -knockout bmdms ( figure 3g ). meanwhile, adp-reduced vsv rna replication was compensated in p2y 13 -deficient pems ( figure 3h) . furthermore, the protein expression of vesicular stomatitis virus glycoprotein (vsv-g) was significantly increased in p2y 13 -deficient pems and overexpression of p2y 13 in hela cells significantly repressed vsv replication ( figure 3i ; supplementary figure 1 ifn signal pathway increases p2y 13 expression. (a) mouse bmdms were treated with ifn-α, ifn-β, or ifn-γ (100 ng/ml) for 2 h, and mrna expression of p2y family was detected by q-pcr. (b) mouse pems were treated with ifn-β (100 ng/ml) for 4 or 8 h, and the expression of p2y 13 protein was detected by western blotting. (c) mouse bmdms were pretreated or not with anti-ifn-α/β receptor igg (10 μg/ml) for 4 h before being treated with ifn-β (100 ng/ml) for 4 h, and the expression of p2y 13 mrna was detected by q-pcr. (d) mouse pems were pretreated or not with ruxolitinib (10 μg/ml) for 1 h and then stimulated with ifn-β (100 ng/ml) for 4 h. the expression of isg15 and p2y 13 mrna was detected by q-pcr. (e) mouse pems were pretreated with or without fludarabine (100 μg/ml) for 30 min and then stimulated with ifn-β (100 ng/ml) for 4 h. the expression of isg15 and p2y 13 mrna was detected by q-pcr. (f) mouse pems were pretreated or not with fludarabine (100 μg/ml) for 30 min and then infected with vsv (moi = 1) for 12 h. the expression of p2y 13 mrna was detected by q-pcr. (g) tlr3 +/+ and tlr3 −/− mouse pems were treated with poly(i:c) (20 μg/ml) for 4 h. the expression of ifn-β, isg15, and p2y 13 mrna was detected by q-pcr. data are shown as mean ± sd. **p < 0.01; ***p < 0.001. figure s2c and d). taken together, our data suggested that p2y 13 deficiency facilitates vsv infection and adp-mediated antiviral activities primarily through the p2y 13 receptor. to further confirm the in vivo antiviral activities of adp/p2y 13 , we challenged wild-type and p2y 13 -deficient mice with a lethal and then infected with vsv (moi = 1) for 12 h. vsv titers in supernatants were measured by plaque assay. (i and j) mouse bmdms were treated with adp (100 μm) for 6 h and then infected with ndv (moi = 0.1) and hsv-1 (moi = 0.1) for 24 h virus rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001. dose of vsv. as shown in figure 4a , the survival of adp-treated wild-type mice was increased obviously, but there was little change in that of p2y 13 -deficient mice. p2y 13 -deficient mice were more susceptible to vsv infection. on the other hand, if we intraperitoneally injected mice with adp to activate p2y 13 , vsv distributions in the liver, spleen, and lung were all significantly decreased ( figure 4b ). similar data were observed during hsv-1 infection ( figure 4c ). besides, more vsv rna replicates were found in p2y 13 -deficient mice liver ( figure 4d ). as a neurotropic virus, vsv can invade the nervous system and is transmitted directly via the transcutaneous or transmucosal route. thus, to mimic the natural infection of vsv, we treated mice with adp through nasal dripping and then intranasally infected with vsv. as shown in figure 4e and f, vsv rna replication and vsv-g in olfactory bulbs were both significantly reduced by adp. taken together, these data demonstrated that adp/p2y 13 plays an important role in protecting mice from vsv infection, suggesting that adp/p2y 13 has potential as a novel antiviral drug target. adp/p2y 13 restricts viral replication by inhibiting camp signaling type i ifn plays pivotal roles in fighting against the invaded virus, so we tested whether it was involved in adp/p2y 13mediated antiviral activities. however, when we treated bmdms with adp, the rna expression of ifn-α and ifn-β was little changed (supplementary figure s3a and b) . p2y 13 signaling can suppress the activity of adcy and the production of camp. to confirm that, raw264.7 cells were pretreated or not with mrs 2211 before being treated with adp. as shown in figure 5a , adp significantly suppressed the production of camp, while p2y 13 antagonist mrs 2211 rescued this phenomenon. adp (50 mg/kg) for 6 h and then intraperitoneally injected with vsv (1 × 10 8 pfu/g). mice were executed for 5 days and mouse survival was recorded for 10 days. (b) six-week-old mice were intraperitoneally treated with adp (50 mg/kg) for 6 h and then intraperitoneally injected with vsv (1 × 10 8 pfu/g) for 24 h. vsv rna replicates in organs were detected by q-pcr. (c) six-week-old mice were intraperitoneally treated with adp (50 mg/kg) for 6 h and then intraperitoneally injected with hsv-1 (1 × 10 7 pfu/g) for 36 h. hsv-1 rna replicates in livers were detected by q-pcr. (d) eight-week-old p2y 13 +/+ or p2y 13 −/− mice were intraperitoneally injected with vsv (1 × 10 8 pfu/g) for 24 h and vsv rna replicates in livers were detected by q-pcr. (e) six-week-old mice were treated with adp (5 mg/kg) through nasal dropping for 6 h and then intranasally infected with vsv (1 × 10 8 pfu/g) for 24 h. vsv rna replicates in olfactory tissues were detected by q-pcr. (f) six-week-old mice were treated as in e, and vsv invaded in olfactory tissues were detected by immunofluorescence. scale bar, 500 μm. data are shown as mean ± sd. **p < 0.01; ***p < 0.001. however, to our surprise, vsv infection caused an obvious increase in cellular camp levels ( figure 5b) . besides, p2y 13 -deficient pems showed higher cellular camp levels than wild-type pems during vsv infection ( figure 5c ). so, we examined the role of camp in adp/p2y 13 -mediated antiviral activities. we first found that adcy activator forskolin blocked adp-mediated the cells were then infected with vsv (moi = 0.01) for 12 h. vsv rna replicates were detected by q-pcr and vsv titers in supernatants were measured by plaque assay. (i) raw264.7 cells were pretreated or not with kh7 (10 μm) for 1 h before being treated with adp (100 μm) for 6 h. the cells were then infected with hsv-1 (moi = 0.01) for 16 h, and hsv-1 rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. antiviral activity completely ( figure 5d and e). furthermore, camp treatment promoted viral replication obviously and also rescued the adp-reduced cell cytopathic effects and vsv replication ( figure 5f and g; supplementary figure s3c and d). consistent with this, kh7 (a selective inhibitor of adcy) treatment significantly inhibited cell cytopathic effects and vsv replication (supplementary figure s3e and f). adp-mediated antiviral activities were also significantly blocked by kh7 ( figure 5h and i). the cell viability assays revealed that these phenomena were not caused by the toxicity of forskolin, camp, and kh7 (supplementary figure s3g-i) . taken together, these results suggested that camp plays a key role in adp/p2y 13 -mediated antiviral activities. adp/p2y 13 restricts viral replication in an epac1-dependent manner when intracellular camp is increased by upstream signaling, epac and protein kinase a (pka) could be activated as classical sensors for camp. thus, we treated hela cells with esi-09 (epac1/2 inhibitor), hjc0350 (epac2 selective inhibitor), and h89 (pka inhibitor) at a safety concentration (supplementary figure s4a -c). as shown in supplementary figure s4d , the replication of vsv is dramatically suppressed by both esi-09 and h89, but not hjc0350, suggesting that epac1 and pka may be involved in adp/p2y 13 -mediated restriction to vsv. however, when we inhibited pka with h89, the replication of vsv ( figure 6a ) and hsv-1 ( figure 6b ) in hela cells are still suppressed by adp, whereas adp-reduced vsv, hsv-1, and ndv replication was significantly blocked by esi-09, but not hjc0350 ( figure 6c -f and supplementary figure s4e) . besides, the adpreduced vsv-g expression was also blocked by esi-09 ( figure 6g) . these data show that epac1 is crucial in adp/p2y 13mediated antiviral activities. in addition, we generated an epac1knockout cell line using the crispr/cas9 system ( figure 6h ). as shown in figure 6i and j, when epac1 is deficient, adp-mediated suppression of vsv rna replication and vsv-g expression were eliminated. taken together, these results demonstrated that adp/p2y 13 -mediated antiviral activities are dependent on epac1. inhibiting epac1 with esi-09 in raw264.7 cells significantly suppressed vsv and hsv-1 replication, and epac1 knockout also inhibited hsv-1 replication, demonstrating that either inhibition or deletion of epac1 has broad-spectrum antiviral activities (supplementary figure s4f-h) . to further confirm the key role of epac1 in viral infection, we detected the expression of epac1 in viral-infected cells. as shown in figure 7a , when infected raw264.7 cells with vsv, ndv, and hsv-1, rna expression of epac1 was increased significantly. deficiency in p2y 13 maintained higher expression of epac1 in pems after infection with vsv ( figure 7b ). in addition, the expression of epac1 protein also upregulated after virus infection as shown in figure 7c and supplementary figure s5a , b. however, to our surprise, the increased expression of epac1 induced by vsv was reduced by adp in a dose-dependent manner (supplementary figure s5c and d) . to exclude that the downregulation of epac1 may be due to the attenuation of vsv replication by adp, cell lines were only treated with adp. as shown in figure 7d -f and supplementary figure s5e , the endogenous rna expression of epac1 was directly inhibited by adp. the protein expression of epac1 was also inhibited by adp ( figure 7g and h). taken together, these data demonstrated that the virusincreased epac1 expression is suppressed by adp, which restricts the viral infection in host cells. p2y 12 and p2y 13 , as membrane receptors for adp, are both gi-coupled receptors that inhibit the activity of adcy and reduce the production of camp (vitiello et al., 2012) . p2y 12 is highly expressed in platelets and plays a central role in platelet activation, which has become a drug target for atherothrombotic diseases, whereas p2y 13 is mainly expressed in the bone marrow, spleen, liver, and brain (savi et al., 2006; vitiello et al., 2012) . previous studies have shown that p2y 12 and p2y 13 are both involved in the development of pain behavior during nerve injury and the expression of pro-inflammatory cytokines (kobayashi et al., 2012; liu et al., 2017) . however, the role of p2y 12 and p2y 13 receptors in viral infection remains unclear. here we demonstrate that p2y 13 is upregulated by type i ifn (p2y 12 is little changed) and its endogenous ligand, adp, is released as danger signals from virusinfected cells. consequently, extracellular adp-activated p2y 13 protects the host against viral infection in a camp-dependent manner. thus, our study reveals an important role of adp/p2y 13 in fighting against viral infection, which demonstrates the potential of purinergic receptors as novel antiviral targets. upon viral infection, the host initiates a series of signaling cascades to induce the production of type i ifn, which results in the induction of isgs to exclude invaded virus (ooi et al., 2014; zhan et al., 2015) . previous studies reported that isgs could further promote the production of type i ifn to eliminate the virus, such as isg15 and ifit3 (lu et al., 2006; liu et al., 2011) . therefore, we tested whether adp/p2y 13 influences the expression of type i ifn. our results show that adp enhanced ifn expression by only a little. because adp clearly inhibited virus replication, we hypothesized that there must be another way for adp/p2y 13 to restrict viral infection. thus, the classic gi-coupled signaling pathway came to mind. interestingly, we found that adp-inhibited vsv replication and cell cytopathic effects were rescued by both adcy activator forskolin and camp. moreover, similar to adp, the adcy inhibitor kh7 also inhibited vsv replication, suggesting that camp plays a key role in adp/p2y 13mediated antiviral activities. as the first identified second messenger, camp plays a crucial role in microbial pathogenesis (mcdonough and rodriguez, 2011) . pka and epac are the two classical sensors for intracellular camp, which are essential for many biological processes (gloerich and bos, 2010) . pka plays important roles in metabolism, cell cycle, cell migration, differentiation, and apoptosis (yan et al., 2017) . previous studies have reported that pka can inhibit ifn induction of the jak/stat pathway (david et al., 1996) . recently, a new study reported that pka catalytic (pkac) figure 6 adp-mediated antiviral activities are dependent on epac1. (a) hela cells were treated or not with h89 (10 μm) for 1 h before being treated with adp (100 μm) for 6 h and then infected with vsv (moi = 0.01) for 12 h. vsv rna replicates were detected by q-pcr. (b) hela cells were treated or not with h89 (10 μm) for 1 h before being treated with adp (100 μm) for 6 h and then infected with hsv-1 (moi = 0.01) for 16 h. hsv-1 rna replicates were detected by q-pcr. (c) hela cells were pretreated or not with esi-09 (10 μm) for 1 h before being exposed to adp (100 μm) for 6 h. the cells were then infected with vsv (moi = 0.01) for 12 h and vsv rna replicates were detected by q-pcr. (d and e) hela cells were pretreated or not with esi-09 (10 μm) for 1 h before being exposed to adp (100 μm) for 6 h. the cells were then infected with hsv-1 (moi = 0.01) or ndv (moi = 0.01) for 16 h and viral rna replicates were detected by q-pcr. (f) raw264.6 cells were treated or not with esi-09 (10 μm) for 1 h before being exposed to adp (100 μm) for 6 h. the cells were then infected with hsv-1 (moi = 0.01) for 16 h, and hsv-1 rna replicates were detected by q-pcr. (g) hela cells were pretreated or not with esi-09 (10 μm) for 1 h before being exposed to adp (100 μm) for 6 h. the cells were then infected with vsv (moi = 0.01) for 12 h and vsv-g protein levels were detected by western blotting. (h) epac1 +/+ and epac1 −/− hela cells were exposed to vsv (moi = 0.01) for 12 h, and the expression of epac1 was detected by western blotting. (i) epac1 +/+ and epac1 −/− hela cells were treated with adp (100 μm) for 6 h and then infected with vsv (moi = 0.01) for 12 h. vsv rna replicates were detected by q-pcr. (j) epac1 +/+ and epac1 −/− hela cells were treated with adp (100 μm) for 6 h and infected with vsv (moi = 0.01) for 12 h. vsv-g protein levels were detected by western blotting. data are shown as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. subunits α and β could negatively regulate rna virus-triggered signaling and ultimately attenuate the innate antiviral response (yan et al., 2017) . epac has two isoforms, epac1 and epac2. the camp/epac signal pathway is important for controlling various cellular functions, including cell adhesion, exocytosis, and microtubule dynamics. previous studies have reported that epac1 is a potential target for the treatment of fatal rickettsioses and that the silencing of epac1 gene expression renders cells resistant to mers-cov infection (gong et al., 2013; tao et al., 2014) . in our study, we found that both pka and epac inhibitors suppress viral replication. however, only epac inhibition could block adp/p2y 13 -mediated antiviral activities. also, we found that the epac2 inhibitor could not block adp/p2y 13 -mediated antiviral activities. these findings suggest that adp/p2y 13 -mediated antiviral activities are mainly dependent on epac1. knockout of epac1 in hela cells showed similar results and further proved this point. taken together, our results showed that, upon viral infection, the virus can trigger an upregulation of intracellular camp levels to activate epac1 and thereby facilitating virus replication. however, the virus also triggers adp released from these cells, which will activate p2y 13 and suppress the camp/ epac1 signal pathway to restrain virus replication. this phenomenon revealed a negative feedback regulation in animals during viral infection, and that activation of p2y 13 and inhibition of the camp/epac1 signal pathway are potential antiviral strategies. p2y 1 , p2y 12 , and p2y 13 knockout mice were described previously (zhang et al., 2018) . tlr3 knockout mice were a gift from professor yuping lai (east china normal university) and were described previously (wu et al., 2016) . c57bl/6 wild-type mice were purchased from the shanghai laboratory animal company. all of the mice were maintained in specific pathogen-free (spf) facilities at the animal center of east china normal university. all animal experiments were done following institutional guidelines. reagents adp, adp-β-s, mrs 2179, forskolin, camp, kh7, esi-09, h89, anti-β-actin antibody, and anti-vsv-g antibody were purchased from sigma-aldrich. ruxolitinib was from targetmol. fludarabine was obtained from calbiochem. polyinosinic-polycytidylic acid [poly(i:c)] was purchased from invivogen. adp-glo™ kinase assay kit was purchased from promega. mrs 2211 and anti-gpr86 (p2y 13 ) antibodies were purchased from abcam. hjc0350 was purchased from selleck. the anti-epac1 antibody was from bioss. primescript rt master mix and sybr premix ex taq were from takara. ifn-α, ifn-β, and ifn-γ were purchased from sino biological inc. the camp elisa kit, mouse ifn-α/β receptor block/neutralize polyclonal goat igg (af1083), and normal goat igg control were from r&d systems. p2y 13 plasmid was obtained from biogot technology, co., ltd. the bmdms were prepared as follows: bone marrow cells were collected from tibias and femurs by flushing with dmem, and the cell suspension was filtered through a 40-μm cell strainer to remove any cell clumps. bone marrow cells were then cultured in dmem containing 10% fbs, 1% penicillin/streptomycin, and 20% l929 culture supernatants. the pems were prepared as follows: mice were intraperitoneally injected with 3 ml of 4% sterile thioglycollate medium and, 3 days later, mice were sacrificed and the peritoneal cavities washed with 10 ml pbs. the cell suspension was filtered through a 70-μm cell strainer to remove any cell clumps. pems were then cultured in dmem containing 10% fbs and 1% penicillin/streptomycin. raw264.7, hek293t, and hela cells were obtained from the american type culture collection and cultured in dmem containing 10% fbs and 1% penicillin/streptomycin. after stimulation, total rna was extracted with rnaiso plus (takara) from the cells. cdna was synthesized from extracted total rna using the primescript rt master mix perfect real time kit (takara) according to the manufacturer's protocol. quantitative real-time pcr was performed using a sybr-green premix (takara). the sequence-specific primers are listed in supplementary table s1 . adp release assay raw264.7 cells, mouse bmdms, and hek293t cells were plated in 24-well plates (2 × 10 5 cells per well) overnight and then infected with viruses for various periods. adp release into the cell culture supernatants was detected using adp-glo™ kinase assay kit according to the manufacturer's protocol. raw264.7 cells were plated in 96-well plates (2 × 10 4 cells per well) overnight. the cells were treated with adp for 6 h and then infected with vsv for 24 h. mts (20 μl, promega) was added to each well. after incubated for 1 h at 37°c, the absorption was measured at 490 nm. cell viability of the untreated group was normalized to 100%. hela and hek293t cells were seeded into 24-well plates (1.5 × 10 5 cells per well) overnight. the cells were treated with adp for 6 h and then infected with vsv-gfp or mlv-gfp for 24 h. cells containing virus were determined after gating on gfp-positive cells. hek293t cells were plated in 96-well plates (8 × 10 3 cells per well) overnight. twelve hours after vsv (moi = 0.01) or hsv-1 (moi = 0.01) infection, cells were fixed with 4% paraformaldehyde in pbs for 20 min at room temperature. after incubation with crystal violet for 30 min, the cells were then washed with pbs five times before the photographs were taken. cells were grown in 6-well plates. after stimulation, cells were lysed with radio immunoprecipitation assay (ripa) buffer supplemented with protease and phosphatase inhibitors. whole-cell lysates were separated by 10% sds-page, transferred to nitrocellulose membranes, and blocked with 5% bsa. immunoblots were hybridized with the respective primary antibodies. protein bands were visualized with the appropriate fluorescent secondary antibodies and the odyssey laser digital imaging system (gene company). cells were infected with viruses for the indicated time and moi. for mouse survival assay, 8-week-old p2y 13 +/+ or p2y 13 −/− mice were intraperitoneally treated with adp (50 mg/kg) or not treated for 6 h and then intraperitoneally injected with vsv (1 × 10 8 pfu/g). executed these for 5 days and mouse survival was recorded for 10 days. for in vivo studies of viral replication, 6to 8-week-old mice were intraperitoneally treated with adp (50 mg/kg) or not treated for 6 h and then intraperitoneally injected with vsv (1 × 10 8 pfu/g) or hsv-1 (1 × 10 7 pfu/g) for the indicated time and viral rna replicates in organs were detected by q-pcr. vero cells were plated in 12-well plates (2 × 10 5 cells per well) overnight. the supernatants from vsv-infected cells were serially diluted and infected vero cells for 1 h. then the supernatant was removed and the cells were covered with dmem containing 1% low-melting point agarose. plaques were counted after 20 h. for immunofluorescence assay, 6-week-old mice were anesthetized with ketamine/rompun and treated with 5 mg/kg adp through nasal dropping for 6 h before being intranasally infected with vsv (1 × 10 8 pfu/g). after 24 h of infection, mice were sacrificed, and the olfactories were fixed with 4% paraformaldehyde in pbs overnight. slices (8-μm) from olfactories were permeabilized with 0.1% triton x-100 and blocked with 5% bsa. tissues were then incubated with anti-vsv-g antibody overnight and alexa-fluor-488-conjugated secondary antibody for 2 h. finally, the tissues were stained with dapi and visualized with a fluorescence microscope. for camp detection, raw264.7 cells or pems were plated in 6-well plates (1 × 10 6 or 2 × 10 6 cells per well, respectively) overnight. after stimulation, the cells were lysed with 200 μl cell lysis buffer and the amounts of camp were measured by elisa (r&d systems) according to the manufacturer's instructions. to generate an epac1-knockout cell line, a crispr/cas9 system was used. the epac1-knockout target sequence used was 5′-aaatgcactccggaccacg-3′. a lenticrisprv2 plasmid containing the epac1-knockout target sequence was transduced into hela cells. three days later, puromycin-resistant single clones were selected, and the epac1-knockout clones were identified by western blotting with the anti-epac1 antibody. all data were analyzed using the graphpad prism software and shown as mean ± sd. the student t-test (two-tailed) was used to analyze the significance of data and p-value <0.05 was considered statistically significant. supplementary material is available at journal of molecular cell biology online. induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation purinergic signaling: a fundamental mechanism in neutrophil activation purinergic p2y 14 receptor modulates stress-induced hematopoietic stem/progenitor cell senescence activation of protein kinase a inhibits interferon induction of the jak/stat pathway in u266 cells the p2x7 receptor in infection and inflammation nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance purinergic signaling during inflammation impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action. microbiol type 1 interferons and the virushost relationship: a lesson in detente epac: defining a new mechanism for camp action exchange protein directly activated by camp plays a critical role in bacterial invasion during fatal rickettsioses errα negatively regulates type i interferon induction by inhibiting tbk1-irf3 interaction nucleotide signalling during inflammation immune cell regulation by autocrine purinergic signalling the role of pattern-recognition receptors in innate immunity: update on toll-like receptors multiple p2y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol ifn-induced tpr protein ifit3 potentiates antiviral signaling by bridging mavs and tbk1 p2y12 and p2y13 receptors involved in adpβs induced the release of il-1β, il-6 and tnf-α from cultured dorsal horn microglia isg15 enhances the innate antiviral response by inhibition of irf-3 degradation the myriad roles of cyclic amp in microbial pathogens: from signal to sword novel antiviral host factor, tnk1, regulates ifn signaling through serine phosphorylation of stat1 mechanisms of type-i-and type-ii-interferon-mediated signalling positional effect of phosphorylation sites 266 and 267 in the cytoplasmic domain of the e2 protein of hepatitis c virus 3a genotype: interferon resistance analysis via sequence alignment receptors for purines and pyrimidines the active metabolite of clopidogrel disrupts p2y12 receptor oligomers and partitions them out of lipid rafts transformation of astrocytes to a neuroprotective phenotype by microglia via p2y1 receptor downregulation adap2 is an interferon stimulated gene that restricts rna virus entry blocking of exchange proteins directly activated by camp leads to reduced replication of middle east respiratory syndrome coronavirus immunoregulation through extracellular nucleotides hyperglycaemia inhibits reg3a expression to exacerbate tlr3-mediated skin inflammation in diabetes pkacs attenuate innate antiviral response by phosphorylating visa and priming it for march5-mediated degradation phosphatase pp4 negatively regulates type i ifn production and antiviral innate immunity by dephosphorylating and deactivating tbk1 two disparate ligand-binding sites in the human p2y1 receptor extracellular adp facilitates monocyte recruitment in bacterial infection via erk signaling this work was supported by the national key r&d program of china (2018yfa0507000 to b.d.), the national natural science conflict of interest: none declared. key: cord-285749-0ejhd9nw authors: hoffmann, markus; krüger, nadine; zmora, pawel; wrensch, florian; herrler, georg; pöhlmann, stefan title: the hemagglutinin of bat-associated influenza viruses is activated by tmprss2 for ph-dependent entry into bat but not human cells date: 2016-03-30 journal: plos one doi: 10.1371/journal.pone.0152134 sha: doc_id: 285749 cord_uid: 0ejhd9nw new world bats have recently been discovered to harbor influenza a virus (fluav)-related viruses, termed bat-associated influenza a-like viruses (batfluav). the internal proteins of batfluav are functional in mammalian cells. in contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (ha)-like (hal) and neuraminidase (na)-like (nal), and these proteins need to be replaced by their human counterparts to allow spread of batfluav in human cells. here, we employed rhabdoviral vectors to study the role of hal and nal in viral entry. vectors pseudotyped with batfluav-hal and -nal were able to enter bat cells but not cells from other mammalian species. host cell entry was mediated by hal and was dependent on prior proteolytic activation of hal and endosomal low ph. in contrast, sialic acids were dispensable for hal-driven entry. finally, the type ii transmembrane serine protease tmprss2 was able to activate hal for cell entry indicating that batfluav can utilize human proteases for hal activation. collectively, these results identify viral and cellular factors governing host cell entry driven by batfluav surface proteins. they suggest that the absence of a functional receptor precludes entry of batfluav into human cells while other prerequisites for entry, hal activation and protonation, are met in target cells of human origin. influenza a viruses (fluav) are enveloped, negative stranded rna viruses that pose a major threat to public health [1] . the ability of fluav to constantly adapt to immune pressure allows these viruses to continuously circulate in the human population, resulting in annual influenza epidemics (seasonal influenza [2, 3] ). infants, children and the elderly are at particular risk of developing severe disease upon infection with seasonal fluav and it has been estimated that world-wide 250,000 to 500,000 people die each year of seasonal influenza [1] . were detached by either resuspension in fresh culture medium (hek-293t cells) or by the use of trypsin/edta (paa laboratories). shuttle vectors harboring codon-optimized (for expression in human cells) open reading frames coding for the published amino acid (aa) sequences of the hal of the two batfluav, a/little yellow-shouldered bat/guatemala/153/2009 (h17/n10) (genbank: cy103876.1, hal17) and a/flat-faced bat/peru/033/2010 (h18/n11) (genbank: cy125945.1, hal18), were purchased from a commercial service (eurofins mwg operon) and cloned into the pcg1 expression vector, that was kindly provided by r. cattaneo, via bamhi and xbai restriction sites. the pcaggs-based expression plasmid for nal of batfluav a/little yellowshouldered bat/guatemala/153/2009 (h17/n10) (genbank: cy103878.1, nal10) was provided by m. schwemmle. hal equipped with a c-terminal flag epitope (dykddddk, hal17-flag and hal18-flag) were constructed by pcr and controlled for sequence integrity by automated sequence analysis. in addition, we used pcaggs-based expression plasmids for the ha and na of a/wsn/33 (h1n1) and a/singapore/1/57 (h2n2) (genbank: ay209895.1) [32, 33] . furthermore, we employed pcaggs-based expression plasmids for the ha of a/south carolina/1/18 (h1n1) (genbank: af117241.1) and the na of a/brevig mission/1/1918 (h1n1) (genbank: af250356.2) that were generated from previously used constructs [32] . the expression plasmid for the glycoprotein (g) of vesicular stomatitis virus (vsv, indiana strain, vsv-g; genbank: aj318514.1) was generated by inserting the vsv-g orf into the pcg1 expression vector and has been used in previous studies [28, 31, 34, 35] . furthermore, expression plasmids for nipah virus fusion protein (f, genbank: af212302) and glycoprotein (g, synthetic, genbank: af212302; derived from niv/my/hu/1999/cdc) were used [36] . for experiments analyzing proteolytic activation of hal17 and hal18 by human type-ii transmembrane serine proteases (ttsps), we employed expression plasmids for tmprss2, tmprss11e (desc-1) and tmprss13 (mspl), which have been described previously [32, 33] . we employed a replication-deficient vsv vector for pseudotyping that contains two separate open reading frames, coding for enhanced green fluorescent protein (egfp) and firefly luciferase (fluc), instead of the genetic information for vsv-g, vsv ã δg-fluc [28, 31, 35] . propagation of vsv ã δg-fluc was performed in a previously described vsv-g-expressing, transgenic cell line [37] . generation of vsv pseudotypes (vsvpp) was performed as follows: hek-293t cells were transfected by calcium-phosphate precipitation with expression plasmids encoding viral surface proteins, vsv-g (positive control) , niv-f/g, fluav-ha and/or na and bat-fluav-hal and/or nal, or empty plasmid (pcaggs) as negative control. in order to investigate the potential of human ttsps to proteolytically activate batfluav-hal for host cell entry, we additionally cotransfected the cells with expression plasmids for tmprss2, desc-1 or mspl. at 16 h post transfection, the cells were inoculated with vsv ã δg-fluc at a multiplicity of infection of 3 for 1 h at 37°c and 5% co 2 . subsequently, the cells were washed and incubated with an anti-vsv serum to neutralize residual input virus. finally, the cells received fresh culture medium and were further incubated for 16-20 h, before the vsvpp-containing supernatants were collected, clarified from cell debris by centrifugation and aliquoted. aliquots were stored at 4°c for a maximum of 7 days. for proteolytic activation of ha/hal by trypsin, pseudotypes were incubated with bovine trypsin (sigma-aldrich; final concentration: 50 μg/ml) for 20 min at 37°c. subsequently, trypsin was inactivated by addition of soybean trypsin inhibitor (sigma-aldrich; final concentration: 50 μg/ml). to investigate the roles of sialic acids and endosomal acidification in batfluav entry, we used recombinantly-produced, bacterial sialidase (clostridium welchii, sigma-aldrich) and ammonium chloride (sigma-aldrich). for treatment, the cell culture supernatant was removed and the cells were washed with phosphate buffered saline (pbs) before culture medium containing water (negative control), ammonium chloride (50 mm) or different concentrations of bacterial sialidase (1.5, 15 or 150 mu) was added. after 2 h of incubation at 37°c and 5% co 2 , the supernatant was removed, the cells washed and then inoculated with pseudotypes, as described below. transduction of cell lines with rhabdoviral pseudotypes and quantification of fluc activity all transduction experiments were performed in 96-well plates using quadruplicate samples. at 24 h post seeding, the cell culture medium was removed and the cells were washed with pbs. the cells were either directly inoculated with vsvpp or treated as specified above and then inoculated. vsvpp inoculation was performed for 1 h at 37°c and 5% co 2 . afterwards, the inoculum was removed and the cells were again washed and incubated with fresh culture medium for 16-18 h at 37°c and 5% co 2 . for the quantification of the fluc activity as an indicator of transduction efficiency, the cell culture supernatant was removed and the cells were washed with pbs. next, 50 μl of 1x luciferase cell culture lysis reagent (promega) in pbs was added to each well and incubated for 30 min at room temperature, before the cell lysate was transferred to a white, opaque-walled 96-well plate (thermo scientific). the measurement of the fluc activity was carried out in a microplate reader, plate chameleon (hidex), using the microwin2000 software (version 4.44, mikrotek laborsysteme gmbh) and fluc substrates from the luciferase assay system (promega) or beetle-juice (pjk) kits. transduction efficiency, represented by fluc activity, was either displayed in counts per second (cps) or as normalized values. to assess expression of hal proteins, we transfected bhk-21 cells that were grown on coverslips with expression plasmids for hal17-flag or hal18-flag using the lipofecta-mine2000 reagent (thermofisher scientific) according to manufacturers' protocol. cells transfected with an empty expression vector served as negative control. at 24 h post transfection, cells were fixed with 4% paraformaldehyde/pbs, permeabilized by incubation with 0.2 m triton x-100/pbs (10 min at room temperature) and subsequently incubated with anti-flag (mouse, 1:1,000, sigma-aldrich) and cy3-labeled anti-mouse (1:750, sigma-aldrich) antibodies. after each antibody incubation, the cells were washed three times with pbs and finally incubated with dapi (roth, 5 min/37°c) to stain the nuclei before the coverslips were mounted on glass slides using mowiol (applichem) supplemented with anti-bleaching reagent (dabco, roth). representative pictures were taken at a 10x magnification using a nikon eclipse ti fluorescence microscope and the nis elements ar software (nikon). batfluav-hal cleavage was investigated by cotransfection of hek-293t cells, which were grown in 6-well plates, with expression plasmids for batfluav-hal and different ttsps (tmprss2, desc-1 or mspl) or by incubation of hal-expressing cells with trypsin (1, 5, 10, 50 μg/ml; 20 min/37°c) directly before cell lysates were produced. cells cotransfected with empty plasmid and not subjected to trypsin treatment served as negative control. the 1918-ha served as positive control, since cleavage by ttsps and trypsin has been previously shown [33] . at 24 h post transfection, the cells were washed with pbs, resuspended in 100 μl 2x sds-containing lysis buffer (50 mm tris [ph 6.8], 10% glycerol, 2% sds, 5% β-mercaptoethanol, 0.1% bromophenol blue, 1 mm edta) and boiled for 20 min at 96°c. to assess incorporation of hal into vsvpp, 1 ml of the respective pseudotypes was loaded onto a 20% sucrose cushion in pbs and centrifuged at 17,000x g for 2 h at 4°c. after discarding the supernatant, the pelleted pseudotypes were mixed with 30 μl 2x sds-containing lysis buffer and boiled for 20 min at 96°c. finally, all samples were subjected to immunoblot analysis. for this, anti-flag (mouse, 1:1,000, sigma-aldrich), anti-fluav (goat, 1:1,000, millipore), anti-vsv-m (mouse, 1:1,000, kerafast), anti-vsv-g (i1, mouse hybridoma supernatant from crl-2700, atcc, 1:200) and anti-ß-actin (mouse, 1:1,000, sigma-aldrich) served as primary antibodies. peroxidase-coupled anti-mouse (1:10,000, dianova) and anti-goat (1:5,000, dianova) antibodies served as secondary antibodies. signal detection was carried out in a chemocam imager together with the chemostar professional software (both intas) using a self-made chemiluminescence substrate (recipe available upon request). in order to assess statistical significance, two-tailed student's t-tests were performed. to test whether hal17 and hal18 are comparably expressed and incorporated into rhabdoviral pseudotypes, both proteins were equipped with a c-terminal flag epitope (hal17-f-lag, hal18-flag), since no batfluav-hal-specific antibody was available. upon transfection of bhk-21 cells similar numbers of hal-expressing cells were detected by fluorescence microscopy ( fig 1a) and the intensity of the fluorescence signals emitted by the cells was comparable, indicating robust expression of both batfluav-hal proteins. in order to assess hal incorporation into rhabdoviral pseudotypes, we pelleted pseudotype preparations through a sucrose cushion and subjected the samples to sds-page and immunoblotting. using antibodies specific for the flag epitope, vsv-g and vsv matrix protein (vsv-m), we found that vsv-g, as expected, as well as both hal17 and hal18 proteins were incorporated into particles, with incorporation of hal17 being more efficient than that of hal18. (fig 1b) . thus, both hal17 and hal18 were robustly expressed and incorporated into vsvpp, allowing their functional characterization. it has been previously reported that hal and nal of batfluav are not compatible with viral spread in the cell lines tested so far [18, 19] , suggesting that these proteins mediate entry into a restricted set of target cells or are even inactive. in order to gain insights into the functional activity of batfluav-hal and nal, we employed rhabdoviral vectors pseudotyped with these proteins. we inoculated cell lines from different host species, including those standardly used for fluav research, as well as cell lines derived from different bat species (table 1) , as they are the natural reservoir for batfluav. we chose bat cell lines known to be susceptible to infection by viruses of different families [28, 29, 31] and previously used to functionally characterize surface glycoproteins of bat-borne viruses [28, 36, 38] . it is well established that fluav-ha depends on proteolytic cleavage by host cell proteases to transit into an active form [12] and it has been previously reported that batfluav can be activated by exogenous trypsin [39] [40] [41] . therefore, we assessed whether trypsin treatment of the pseudotypes impacts transduction efficiency. as controls, we included the surface glycoprotein(s) of well-characterized fluav strains, a/wsn/33 (h1n1) (wsn-ha, wsn-na), a/south carolina/1/18 we found that none of the human, simian and canine cell lines was susceptible to entry driven by batfluav surface proteins (fig 2a) . in contrast, pseudotypes bearing vsv-g, niv-f/g or wsn-ha/na could readily enter these cells, whereas pseudotypes that harbored 1918-or h2n2-ha/na required activation by exogenous trypsin for efficient transduction (fig 2a) . these results are in agreement with expectations, since activation of wsn-ha is known to be independent of trypsin [42] [43] [44] , although viral infectivity can be enhanced by trypsin treatment. when we focused on bat-derived cell lines, vsv-g, niv-f/g and wsn-ha/na again permitted pseudotype entry without prior trypsin treatment, while pseudotypes harboring 1918-ha/na or h2n2-ha/na were only able to transduce some of the bat cell lines after incubation with trypsin ( fig 2b) . cpkd cells remained refractory to entry mediated by 1918-ha/na or h2n2-ha/na but also showed the lowest susceptibility to all other tested pseudotypes. notably, three bat cell lines (eidni/41, hypni/1.1 and eponi/22.1) were susceptible to entry of pseudotypes bearing hal and nal of batfluav (fig 2b) , demonstrating that surface glycoproteins of batfluav can mediate cellular entry. entry into the three bat cell lines was robust (1-3 log units above the threshold) and required prior treatment of pseudotypes with trypsin, which presumably resulted in the proteolytic activation of hal. furthermore, pseudotypes bearing hal17/nal10 or hal18/nal10 were both able to enter hypni/ 1.1 and eponi/22.1 cells while eidni/41 cells could only be transduced by pseudotypes bearing hal18/nal10 (fig 2b) , suggesting that batfluav of the hl17nl10 and hl18nl11 subtype might exhibit subtle differences in entry efficiency or cell tropism. finally, hal-proteins with a c-terminal flag tag facilitated host cell entry, although with somewhat reduced efficiency as compared to their untagged counterparts, indicating that the proteins used to study hal expression and virion incorporation (fig 1) were functional (data not shown). taken together, we showed that batfluav surface proteins can mediate entry into certain bat cell lines. for further studies on the entry process, we focused on eponi/22.1 cells since they showed the highest susceptibility to entry driven by batfluav surface proteins. the na proteins of human fluav facilitate release of progeny particles from infected cells by removing sialic acids from the cell surface. to study the impact of the batfluav-nal on transduction efficiency, we produced pseudotypes bearing batfluav-hal, -nal or both proteins. for comparison, we generated pseudotypes harboring wsn-ha, wsn-na or both proteins. these pseudotypes were then used for inoculation of mdck (inoculated with pseudotypes bearing wsn proteins) and eponi/22.1 (inoculated with pseudotypes bearing wsn or batfluav proteins) cells. pseudotypes harboring only wsn-na were not able to transduce target cells (fig 3) while pseudotypes bearing either wsn-ha alone or in combination with wsn-na transduced both mdck and eponi/22.1, as expected. transduction efficiency was 500-1,500-fold higher when wsn-na was expressed in cells used for pseudotype production, in keeping with the findings that the presence of na is required for efficient release of ha-bearing vectors and infectious fluav [32, 45, 46] . pseudotypes harboring nal were not infectious while pseudotypes bearing hal robustly transduced eponi/22.1 cells, indicating that batfluav-hal, like wsn-ha, is sufficient to mediate host cell entry. however, unlike wsn-na, the expression of nal in pseudotype producer cells did not increase transduction efficiency of hal-harboring pseudotypes (fig 3) , suggesting that nal is not required for release and/or infectivity of hal containing particles, at least in the experimental system chosen. batfluav-hal does not use sialic acids for host cell entry fluav employ alpha-2,3-(avian viruses) and alpha-2,6-linked (human viruses) sialic acids as receptors for host cell entry [47] [48] [49] [50] [51] . in order to assess the potential role of sialic acids in haldriven entry, we pre-treated the cells with escalating doses of bacterial neuraminidase before transduction. neuraminidase treatment of eponi/22.1 cells reduced transduction mediated by the fluav-ha proteins, as expected (fig 4) . in contrast, sialidase treatment had no effect on pseudotype entry mediated by batfluav-hal, niv-f/g or vsv-g. moreover, pre-treatment of eponi/22.1 cells at the highest dose (150 mu) even enhanced transduction driven by hal and vsv-g (fig 4) . these results indicate that hal does not use sialic acids for host cell entry and suggest that removal of sialic acids might even increase batfluav infectivity, potentially by increasing accessibility of a cellular receptor. endosomal low ph triggers fluav-ha for membrane fusion. therefore, we investigated whether increasing the endosomal ph in eponi/22.1 cells by ammonium chloride treatment impacts hal-driven entry. as expected, ammonium chloride treatment led to a decrease in transduction efficiency mediated by pseudotypes bearing the ha-proteins of fluav of the h1n1 and h2n2 subtype and vsv-g (fig 5) . in contrast, pseudotype entry orchestrated by niv-f and -g was unaffected, again in keeping with published data [52, 53] . finally, haldriven entry was markedly reduced by ammonium chloride, demonstrating that the membrane fusion activity of batfluav-hal is triggered by acidification (fig 5) . fluav-ha is synthesized as an inactive precursor and requires activation by host cell proteases to be responsive to low ph, the trigger for ha-driven membrane fusion [12] [13] [14] . members of the ttsp family activate fluav-ha in cell culture [33, [54] [55] [56] [57] [58] and tmprss2 was previously shown to be essential for fluav-ha activation and viral spread in mice [59] . therefore, we asked whether ttsps able to activate fluav-ha can also activate batfluav-hal. for this, we first investigated batfluav-hal cleavage by tmprss2, desc-1 and mspl, and compared it to cleavage by trypsin. cleavage of the 1918-ha served as positive control. we found that 1918-ha was efficiently processed by all proteases tested, as expected. moreover, we could show that coexpression of tmprss2, desc-1 and mspl, and trypsin treatment resulted in cleavage of the hal precursor (hal 0 ) determined by the appearance of bands corresponding to the hal 2 subunit (fig 6a) . while hal18 was comparably cleaved by all tested ttsps, hal17 cleavage by tmprss2 was more pronounced than proteolysis by desc-1 and mspl (fig 6a) . moreover, hal18 was generally more sensitive to cleavage by ttsps than hal17 (fig 6a) . in order to assess whether batfluav-hal cleavage by ttsps also leads to hal activation for host cell entry, we produced pseudotypes harboring batfluav-hal (hal17 or hal18) in the presence of tmprss2, desc-1 and mspl. as a control, pseudotypes bearing 1918-ha and -na were included in this experiment. the pseudotypes were treated with trypsin to activate ha/hal or were mock-treated before addition to eponi/22.1 cells. pseudotypes bearing 1918-ha and -na and produced in the presence of tmprss2, desc-1 and mspl or treated with trypsin robustly transduced target cells ( fig 6b) . in contrast, infectivity of fluav-ha pseudotypes produced in the absence of ttsps or not treated with trypsin was in the background range (fig 6b) . similarly, batfluav-halbearing pseudotypes were activated by trypsin or ttsps, including tmprss2 (fig 6b) . however, differences in the activation of hal17 and hal18 were observed and correlated with the efficiency of hal protein cleavage, as determined above (fig 6a) . thus, expression of tmprss2 but not desc-1 and mspl conferred robust infectivity to hal17-bearing pseudotypes while all proteases were able to efficiently activate hal18. moreover, transfection of escalating amounts of tmprss2-encoding plasmids increased infectivity of hal17-bearing pseudotypes in a concentration-dependent manner. in contrast, transfection of even the lowest amount of tmprss2 plasmid was sufficient to confer maximal infectivity of hal18bearing pseudotypes, confirming that the efficiency of tmprss2-mediated activation of hal is subtype specific (fig 6c) . in sum, proteolytic activation of batfluav-hal is critical for hal-driven cell entry and proteases able to activate ha can also activate hal. host cell entry by bat-associated influenza viruses the identification of two new fluav, subtypes h17n10 (hl17nl10) and h18n11 (hl18nl11), in new-world bats [16, 17] suggests that bats could serve as a natural reservoir of fluav [16, 17] . unraveling the zoonotic potential of batfluav is of great importance since fluav are major human pathogens, responsible for influenza epidemics and pandemics. while the batfluav replication machinery appears to be functional in different mammalian (including human) cells [16, [18] [19] [20] [21] 60] , reassortment with fluav and flubv is unlikely [18, 19] . regarding batfluav tropism of the viral surface proteins, hal and nal, only human proteases that activate fluav-ha for cell entry also activate batfluav-hal. (a) hek-293t cells were transfected with plasmids encoding ha or hal proteins and either trypsin treated or cotransfected with plasmids encoding type ii transmembrane serine proteases. transfection of empty vector served as negative control. cleavage of ha/hal proteins was analyzed by sds-page and western blotting, employing antibodies against fluav-ha (α-fluav) and the flag epitope (α-flag). detection of ß-actin served as loading control. signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. the results were confirmed in a separate experiment. to assess proteolytic activation of ha/hal proteins, vesicular stomatitis virus-based pseudotypes (vsvpp) were produced in cells transfected to express the indicated type ii transmembrane serine proteases (b) or different amounts of tmprss2 (c). pseudotypes were either directly used for transduction of eponi/22.1 cells (black bars) or previously treated with trypsin (white bars). at 24 h post inoculation, transduction efficiency was measured by quantification of the activity of vsvpp-encoded luciferase in cell lysates. for normalization, transduction by ha-or hal-bearing pseudotypes that were produced in the absence of type ii transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. the result of a single representative experiment carried out with quadruplicate samples is presented. similar results were obtained in three independent experiments carried out with separate pseudotype preparations. error bars indicate standard deviations. a two-tailed, unpaired student's t-test was used to test statistical significance (* = p < 0.05). doi:10.1371/journal.pone.0152134.g006 host cell entry by bat-associated influenza viruses limited information is available, which indicate that batfluav do not engage with canonical fluav receptors [17, [22] [23] [24] . however, until very recently no proof of functional activity of batfluav surface proteins was available [17, [22] [23] [24] [25] [26] [27] . here, we employed a vector system to analyze batfluav-hal and -nal. we show that hal mediates entry into certain bat cell lines and that entry does not depend on the presence of sialic acids on the cell surface. moreover, we demonstrate that nal is not required for production of infectious hal-bearing particles, at least under the conditions examined. finally, our studies revealed that trypsin and ttsps activate hal for host cell entry. we used a vsv-based vector system to study cellular entry of hal and nal-bearing particles. vsv pseudotypes allow convenient analysis of entry driven by diverse glycoproteins [28, 31, 61] , although one should keep in mind that due to differences in particle shape and efficiency of glycoprotein incorporation pseudotypes might not adequately mirror all aspects of cellular entry of authentic viruses [62, 63] . we found that cell lines frequently used for fluav propagation were not susceptible to transduction by hal and nal bearing particles, which is in agreement with the finding that replacement of batfluav-hal and -nal by their fluav counterparts is required for spread of batfluav in the cell lines studied so far [18, 19] . in contrast, inoculation of bat cell lines originating from five different species of micro-and megachiropteran bats revealed that three cell lines, eidni/41, hypni/1.1, eponi/22.1, were susceptible to entry mediated by batfluav surface proteins. eponi/22.1 cells showed the highest susceptibility and were thus used for further studies, while eidni/41 cells were found to be robustly susceptible only to transduction by pseudotypes harboring the hal18. collectively, these findings suggest that batfluav surface proteins can mediate entry into certain bat cells and that entry efficiency might differ between batfluav subtypes. our finding that certain bat cell lines are susceptible to pseudotypes harboring hal of batfluav are in line with observations very recently documented by maruyama et al. who found that out of a diverse panel of bat cell lines tested, cells from miniopterus fuliginosus, miniopterus schreibersii and pteropus giganteus were susceptible to ph-dependent, hal-driven entry [64] . a cell line derived from eidolon helvum spleen cells was found to be non-susceptible in contrast to our findings with a kidney cell line established from the same species, suggesting that receptor expression might differ between organs. somewhat more surprising, maruyama and colleagues also observed hal-driven entry into mdck cells [64] , which was not detected in the present study, and these discrepant results might be attributed to use of mdck cells from different sources or to differences in the method used to quantify pseudotype entry. finally, it is noteworthy that cell lines from bats inhabiting different geographical locations (africa, asia, europe) were found to be susceptible to hal-driven entry, suggesting that entry is not a bottleneck for spread of batfluav between bat species. the finding that batfluav surface proteins can facilitate entry into bat-derived target cells allowed us to investigate which viral and cellular components contribute to the entry process. we first focused on nal. the expression of this protein, unlike expression of na, in pseudotype-producing cells did not increase the titers of vectors harboring the corresponding bat-fluav-hal protein. however, this finding does not exclude that nal, like the na of fluav, acts as a receptor-destroying enzyme. thus, hek-293t cells used for pseudotype preparation were not susceptible to hal-driven entry, most likely because they do not express the appropriate receptor. it thus remains to be analyzed whether batfluav-nal increases release of hal-bearing vectors and authentic batfluav from susceptible bat cell lines. these endeavors might be challenging since transfection of bat cell lines by calcium-phosphate precipitation and liposome-based reagents was inefficient (not shown). the fluav-ha is sufficient to mediate viral binding and entry into target cells and our findings indicate that the same applies to batfluav-hal. in contrast to fluav-ha, however, hal does not depend on the presence of sialic acids for entry. thus, treatment of eponi/22.1 cells with sialidase did not decrease hal-mediated pseudotype entry. these findings are in keeping with the work by maruyama et al. [64] and with structural data indicating that hal does possess a distorted putative sialic acid binding site [17, 24] . contrarily, high amounts of sialidase increased entry efficiency, potentially by increasing access to the elusive receptor. in addition, removal of sialic acids might increase electrostatic interactions of batfluav-hal with cell surface factors, since sialic acids are negatively charged. although hal-driven entry was independent of sialic acids, it did require endosomal acidification (in accordance with maruyama et al. [64] ), which is known to trigger the membrane fusion activity of ha. most likely, protonation also triggers bat-fluav-hal for membrane fusion. however, it cannot be disregarded that the inhibitory effect of ammonium chloride was due to blockade of ph-dependent endosomal cysteine proteases, which activate the surface proteins of several coronaviruses and ebolaviruses [65] [66] [67] [68] . cleavage-activation of fluav-ha by host cell proteases is essential for fluav infectivity. several ttsps can cleave and activate ha in cell culture and recent studies demonstrated that tmprss2 is essential for fluav spread in mice [32, 33, 58, 59] . moreover, polymorphisms in tmprss2 were shown to impact severity of influenza in humans [69] . treatment of bat-fluav-hal-expressing cells with trypsin led to proteolytic cleavage of hal and exposure of hal-bearing pseudotypes to trypsin was required for efficient transduction of target cells, indicating that proteolytic processing is also required for hal function. moreover, coexpression of batfluav-hal with tmprss2, desc-1 or mspl resulted in proteolytic cleavage of hal and rendered the particles infectious in the absence of trypsin treatment, suggesting that bat-fluav-hal can utilize human proteases for their activation. finally, titration of the amounts of tmprss2 had differential effects on the proteolytic cleavage of hal17 and hal18 and on infectivity of pseudotypes bearing these proteins, hinting towards subtle differences in the efficiency of tmprss2 use by these subtypes. whether bat tmprss2 is also able to cleave and activate batfluav-hal remains to be investigated. collectively, our results are most compatible with a scenario in which human cells allow for batfluav-hal activation and triggering but fail to express a receptor, which can be employed by hal for host cell entry. these results, jointly with the documented observation that the batfluav replication machinery is functional in human cells [16, 18, 19] suggest that hal adaptation to a human receptor might be the major hurdle batfluav need to overcome to spread in humans. it will thus be highly interesting to identify the nature of this receptor and its interface with batfluav-hal. of note, during the preparation of this manuscript, maruyama and colleagues published a manuscript reporting batfluav-hal-driven entry into bat cell lines different from those used in the present study (maruyama et al., 2015, doi: 10 .1016/j.virol.2015.11.002.). both studies show that hal-driven entry requires prior proteolytic hal-activation by trypsin and endosomal acidification but is independent of sialic acids. the present work extends these findings by demonstrating that hal can utilize the human enzyme tmprss2 for its activation. seasonal) fact sheet no. 211: world health organisation global epidemiology of influenza: past and present. annual review of medicine the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza 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endosomal compartment activation of the nipah virus fusion protein in mdck cells is mediated by cathepsin b within the endosome-recycling compartment novel type ii transmembrane serine proteases, mspl and tmprss13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication cleavage activation of the human-adapted influenza virus subtypes by matriptase reveals both subtype and strain specificities hat, and tmprss2 activate the hemagglutinin of h9n2 influenza a viruses influenza virus activating host proteases: identification, localization and inhibitors as potential therapeutics proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium tmprss2 is essential for influenza h1n1 virus pathogenesis in mice the n-terminal domain of pa from batderived influenza-like virus h17n10 has endonuclease activity severe fever with thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use dc-sign as a receptor for ph-dependent entry into human and animal cell lines lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins pseudotyping viral vectors with emerging virus envelope proteins characterization of the glycoproteins of bat-derived influenza viruses endosomal proteolysis of the ebola virus glycoprotein is necessary for infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry protease-mediated entry via the endosome of human coronavirus 229e endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry identification of tmprss2 as a susceptibility gene for severe 2009 pandemic a(h1n1) influenza and a(h7n9) influenza. the journal of infectious diseases we would like to thank c. drosten, m. a. müller and m. schwemmle for cell lines and expression plasmids. furthermore, we thank e. berger and i. nehlmeier for excellent technical support. key: cord-004126-u6ts87ur authors: furuyama, wakako; reynolds, pierce; haddock, elaine; meade-white, kimberly; quynh le, mai; kawaoka, yoshihiro; feldmann, heinz; marzi, andrea title: a single dose of a vesicular stomatitis virus-based influenza vaccine confers rapid protection against h5 viruses from different clades date: 2020-01-10 journal: npj vaccines doi: 10.1038/s41541-019-0155-z sha: doc_id: 4126 cord_uid: u6ts87ur the avian influenza virus outbreak in 1997 highlighted the potential of the highly pathogenic h5n1 virus to cause severe disease in humans. therefore, effective vaccines against h5n1 viruses are needed to counter the potential threat of a global pandemic. we have previously developed a fast-acting and efficacious vaccine against ebola virus (ebov) using the vesicular stomatitis virus (vsv) platform. in this study, we generated recombinant vsv-based h5n1 influenza virus vectors to demonstrate the feasibility of this platform for a fast-acting pan-h5 influenza virus vaccine. we chose multiple approaches regarding antigen design and genome location to define a more optimized vaccine approach. after the vsv-based h5n1 influenza virus constructs were recovered and characterized in vitro, mice were vaccinated by a single dose or prime/boost regimen followed by challenge with a lethal dose of the homologous h5 clade 1 virus. we found that a single dose of vsv vectors expressing full-length hemagglutinin (hafl) were sufficient to provide 100% protection. the vaccine vectors were fast-acting as demonstrated by uniform protection when administered 3 days prior to lethal challenge. moreover, single vaccination induced cross-protective h5-specific antibodies and protected mice against lethal challenge with various h5 clade 2 viruses, highlighting the potential of the vsv-based hafl as a pan-h5 influenza virus emergency vaccine. influenza a viruses, which belong to the family orthomyxoviridae, have a single-stranded negative-sense rna genome consisting of eight segments. 1 they are important zoonotic pathogens, with high morbidity in pigs, horses, poultry, and humans. 2 influenza a viruses have two envelope glycoproteins (gps), hemagglutinin (ha) and neuraminidase (na), and are divided into subtypes based on antigenicity. subtypes h1-16 ha and n1-9 na have been isolated from water birds, the natural reservoir of influenza a viruses. 3, 4 until 1997, avian influenza a viruses were considered unlikely to be transmitted directly to humans because they do not bind the human sialic acid-α2,6-galactose (saα2,6gal) receptor with high affinity. 5 however, highly pathogenic avian influenza (hpai) viruses can be transmitted from wild birds upon close contact causing sporadic outbreaks in domestic poultry. this happened for the first time in 1997 in hong kong when 18 human cases of respiratory illness, including six fatalities, were caused by hpai subtype h5n1 viruses. [6] [7] [8] since then, 860 human cases, with 454 deaths (~53% case fatality rate), have been reported by the world health organization. 9 furthermore, some reassortant h5 viruses with different na subtypes (e.g. h5n2, h5n8, and h5n6) originated from the same ancestral h5n1 virus, and have recently emerged in china and spread to other countries in eurasia and north america. [10] [11] [12] [13] [14] [15] since some hpai viruses are resistant to the currently available treatment options for influenza a virus infections namely oseltamivir, amantadine, and interferon (ifn), 16, 17 the development of vaccines is an ongoing effort of high priority for public health to be prepared for a potential epidemic or pandemic of hpai. several different vaccination strategies have been developed against influenza a viruses including inactivated whole virus, liveattenuated influenza virus, viral vectors, and dna vaccines. 18 currently, the fda-approved and licensed whole virus and liveattenuated vaccines against human influenza a viruses are mainly produced in embryonated chicken eggs and the manufacturing process can take up to 9 months. [18] [19] [20] unfortunately, the high pathogenicity of hpai viruses for the chicken embryo reduces virus growth complicating efforts to obtain quality allantoic fluid with high virus titers. therefore, hpai viruses are not suitable as seed viruses for inactivated virus-based vaccine production. vesicular stomatitis virus (vsv) is a single-stranded negativesense rna virus in the family rhabdoviridae. although vsv can cause disease in livestock and other animals, it is highly restricted by the human ifn response and generally does not cause any or only very mild disease. 21 the vsv platform used here is based on the attenuated replication-competent vaccine that produces a rapid and robust immune response to foreign antigens after a single immunization and has been shown to protect against numerous pathogens. [22] [23] [24] [25] [26] especially, the vsv-based ebola virus (ebov) vaccine, vsv-ebov (also known as rvsv-zebov or ervebo), which expresses the ebov gp instead of the vsv gp, is considered safe and highly immunogenic based on data from multiple clinical trials. 27, 28 noteworthy, vsv-ebov has shown promising efficacy against ebov in a phase iii clinical trial 28 and is currently being used in the democratic republic of the congo during the ongoing ebov outbreak. 29 the promising safety profile of this liveattenuated vaccine and the favorable immune cell targeting mediated by the ebov gp makes vsv-ebov an interesting platform for vaccine development. 30 the feasibility of this concept has previously been demonstrated in preclinical studies with vaccines for influenza (hpai virus), flavi-(zika virus), and bunyaviruses (andes virus). 24, [31] [32] [33] in this study, we designed and tested different vsv-ebov-based vaccine vectors expressing different versions of the h5n1 ha (a/vietnam/1203/2004 (vn/1203)) to demonstrate the feasibility of the platform for a fast-acting pan-h5 vaccine. mice were vaccinated with a single dose or prime/boost regimen of the different vaccine candidates and challenged with a lethal dose of homologous h5n1 virus. we found that a single vaccination with vsv-vectors expressing the full-length ha (hafl) induced crossreactive h5-specific antibodies and conferred complete protection against lethal challenge with various h5 clade 2 viruses. furthermore, a single dose of these vaccine vectors provided uniform protection in mice against lethal h5n1 challenge within 3 days after vaccination. we generated vsv-based h5 vaccine vectors by inserting either the full-length open reading frame (orf) of the h5n1 hafl (vn/ 1203) or a soluble version of this gene lacking the transmembrane and cytoplasmic domains but carrying a mutated single-basic cleavage site to prevent cleavage in the cells and a gcn4 leucine zipper domain (shazip) for stabilization of the trimeric structure into the vsv-ebov vector 34, 35 (fig. 1a) . this shazip antigen has previously been shown to be protective in chickens as a subunit vaccine. 34 we also generated a vsv vector expressing the h5n1 hafl alone without the ebov gp (vsv-hafl; fig. 1a ) 36 in order to control for the contribution of the ebov gp to vaccine efficacy. expression of the different h5 antigens from the vsv vectors was confirmed by subjecting the supernatant of infected cells to sds-page and immunoblotting (fig. 1b, supplementary fig. 1 ). first, we showed the presence of vsv particles by detecting the vsv matrix (m) protein in the supernatant of infected cells (fig. 1b, supplementary fig. 1 ). the incorporation of ebov gp into vsv particles differed among the vectors and was, as expected, highest for vsv-ebov for which it is the only surface protein and antigen encoded by this vector (fig. 1b, supplementary fig. 1 ). expression of shazip was verified by detecting the non-cleaved sha 0 precursor likely secreted from infected cells. hafl expression was demonstrated by detecting the furin-cleaved fragment ha 1 in mature spikes on vsv particles. as expected, the incorporation of hafl into recombinant vsv particles was much stronger for vsv-hafl compared to vsv-ebov-hafl, likely because it is the only surface gp and encoded antigen in the vsv vector. next, we performed a series of studies measuring the rate and extent of vaccine virus growth over time. vero e6 cells were infected in triplicate with each vsv-based vector (multiplicity of infection (moi) 0.01) and samples were collected from the supernatant at 24, 48 and 72 h for titration. wild-type vsv (vsvwt) grew more rapidly and to significantly higher titers early post infection compared to any of the other recombinant vsv-based vectors (fig. 1c) . we did not observe any significant difference in the growth kinetics of vsv-based vectors expressing either one (vsv-ebov, vsv-hafl) or two foreign antigens (vsv-ebov-shazip, vsv-ebov-hafl) with most vectors reaching peak titers between 10 7 and 10 8 tcid 50 /ml at 48 h suggesting that the expression of a second antigen did not significantly further attenuate vsv-ebov (fig. 1c) . 50 (400 tcid 50 ) of hpai h5n1. as expected, control and vsv-ebov vaccinated mice succumbed to the lethal h5n1 challenge within 9 days (fig. 2) . single-dose vaccination of the vsv-ebov-shazip showed only 12.5% protection against h5n1 infection with severe weight loss (fig. 2 , left panels). prime/boostvaccination improved the outcome of the vsv-ebov-shazip resulting in mild disease as evidenced by temporary weight loss and moderate disease with 75% survival for vsv-ebov-shazip (fig. 2 , right panels). in contrast, single and prime/boost vaccination with vsv-ebov-hafl or vsv-hafl protected 100% of the mice from lethal challenge with no signs of clinical disease (fig. 2) . in order to improve the protective efficacy of the vsv-shazip vector, we wanted to increase the antigen expression levels. therefore, the shazip or sha (without trimerization domain) antigens were inserted further upstream into the vsv-ebov backbone resulting in two additional vaccines, vsv-shazip-ebov and vsv-sha-ebov (supplementary fig. 2a ). these vaccine viruses were recovered and antigen expression was confirmed in the cell supernatant similarly to the previously generated vaccines ( supplementary fig. 1 ). in vitro growth kinetics demonstrated no difference in comparison to the other vsv constructs (fig. 1b, supplementary fig. 2b ). next, we analyzed the protective efficacy of these improved vsv-sha-ebov vaccine candidates in mice. single-dose and prime/boost-vaccinations with the vsv-shazip-ebov revealed similar protective efficacies compared to vsv-ebov-shazip (fig. 2, supplementary fig. 2c ). interestingly, the h5n1 challenge of vsv-sha-ebov-vaccinated mice demonstrated higher survival rates compared to shazip expressing vsv vectors ( fig. 2, supplementary fig. 1d ). taken together, the challenge experiments demonstrated that hafl is the superior antigen to any of the sha versions as a single dose results in uniform protection using the vsv platform. interestingly, sha performed better than shazip. analysis of vsv-based vaccine-mediated antibody responses total anti-ha (h5) immunoglobulin g (igg) and neutralizing antibody responses from all vsv-vaccinated mice were analyzed in serum samples collected directly prior to challenge (day 0) and day 6 and day 28 post challenge. enzyme-linked immunosorbent assay (elisa) was performed to determine total anti-ha igg levels in the serum of the mice over time (fig. 3 , supplementary fig. 3 ). in control and vsv-ebov-vaccinated mice, we observed no antibody responses on day 0, but ha-specific igg was detected on day 6 after h5n1 challenge with all animals succumbing to infection by day 9 (fig. 3, supplementary fig. 3 ). all haflvaccinated mice responded with ha-specific antibody responses to a single dose on day 0 (fig. 3 , top left panel) that were lower compared to those of the corresponding prime/boost vaccinated mice (fig. 3 , top right panel). the same was observed for sha/ shazip-vaccinated mice ( supplementary fig. 3 ). vsv-hafl prime/ boost vaccination elicited the highest ha-specific igg responses that were significantly higher compared to control mice and the mice vaccinated with vsv-ebov, vsv-shazip-ebov, and vsv-ebov-shazip. in all vaccinated and surviving mice, the h5n1 challenge served as a boost as documented by the increase in haspecific igg titers measured on day 28 post challenge ( fig. 3 , supplementary fig. 3 ). time to immunity of the vsv-based hafl vaccines finally, we determined the minimum time to immunity for the two most promising vaccine candidates, vsv-ebov-hafl and vsv-hafl. groups of eight female balb/c mice were im vaccinated with 1 × 10 4 pfu of vsv-ebov-hafl or vsv-hafl on days 7, 3, or 1 prior to lethal homologous h5n1 challenge. we found that both vaccines resulted in 100% protection with no or little weight loss when mice were vaccinated at least 3 days prior to challenge, whereas vsv-ebov-vaccinated mice succumbed to infection within 10 days (fig. 4) . furthermore, the day −1 vaccinations resulted in partial survival with 62.5% for vsv-hafl and 75% for vsv-ebov-hafl (fig. 4) . overall, the data demonstrate that both vaccine candidates are equally potent inducers of rapid protection with a slight but not statistically significant benefit of vsv-ebov-hafl over vsv-hafl. cross-protection with a single dose of the vsv-based hafl vaccines due to frequently occurring antigenic changes with influenza viruses, it is important to determine if vaccine candidates elicit antibodies against viruses from different antigenic clades within the same subtype. therefore, we performed hemagglutinin inhibition (hi) tests to examine the ability of the vsv-based h5n1 vaccines to generate cross-neutralizing antibody responses against heterologous h5 influenza viruses. for this, we used the day 0 mouse serum samples and a panel of nine attenuated candidate vaccine influenza viruses encoding has belonging to different h5 clades that were isolated from geographically distinct locations (supplementary table 1 ). we found that prime/boost vaccination with vsv-ebov-hafl or vsv-hafl elicited crossneutralizing antibodies against all tested clades (table 1) . crossneutralizing antibodies were also detected in the single-dose vaccination group of these two vaccines; however, and similar to the total ha-specific igg, levels were lower and crossneutralization was not detected for all clades (table 1 ). in contrast, all other vaccines expressing the sha/shazip antigen revealed no cross-neutralizing activities after administration of a single-dose and limited cross-neutralizing activity after the prime/boost. these results demonstrated that the vsv-based vaccines expressing hafl induce more potent cross-neutralizing antibodies than the sha/ shazip antigens. in order to support the cross-protective potential of the vsvbased hafl vaccines, we vaccinated groups of mice with a single dose of 1 × in contrast, all mice vaccinated with vsv-ebov-hafl or vsv-hafl were completetly protected aginast lethal challenge (fig. 5 ), but mice challenged with a/indonesia/5/2005 showed minor body weight loss on days 4-6 after challenge (fig. 5c) . taken together, the vsv vectors expressing hafl confer h5 cross-protection in the mouse model. the 1997 outbreak of human h5n1-caused disease in hong kong was controlled with the depopulation of poultry. 37 however, while this outbreak was contained, hpai h5n1 viruses have been circulating in poultry for almost two decades now and have spread to more than 60 countries. 38 the broad geographic distribution of hpai h5n1 viruses and the risk of transmission to humans causing severe pneumonia with high case fatality rates 39 are a major concern to animal and human health since years. treatment is an option for individual human cases but if these hpais gain transmissibility for humans, vaccines are likely the only public health measure to fight an epidemic or potential pandemic. in this study, we used the well-characterized vsv-ebov vaccine as our starting platform as it has advantages over other vaccine approaches such as ease of genetic modification, efficient and cost-effective manufacturing, proven human safety and immunogenicity profile, and potential favorable immune cell targeting. 27, 28 to define a more optimized vaccine approach, we generated several different vsv-ebov-based vaccine vectors and compared the protective efficacy against hpai h5n1 virus challenge in the mouse model to a vsv-hafl vector without the ebov gp. despite promising results from previous studies in chickens showing that adjuvanted subunit vaccines consisting of the trimeric h5 sha (shazip) induced high levels of cross-neutralizing antibodies (clade 1 and 2.3.4), 34,35 we could not demonstrate convincing protection with the shazip-expressing vsv vectors in this study (fig. 2, supplementary fig. 2c ). in fact, vsv-sha-ebov without the trimerization sequence performed better than the shazipexpressing vectors with complete protection following prime/ boost vaccination (supplementary fig. 2c) . in contrast to all the sha-based vaccines, single doses of the vsv-ebov-hafl or vsv-hafl vectors were sufficient to provide complete protection from lethal homologous h5n1 challenge in mice (fig. 2) . thus, in our study, the vsv vectors expressing hafl are superior over those expressing sha or shazip. it should be noted that vaccine doses in this study were about 100 to 1000 times lower than those used in previous vsv-based hpaiv h5n1 vaccine studies. 32, 36, 40 this is an important observation, as lower-dose vaccination would likely reduce potential adverse effects of vaccination as has been reported ocassionally from human clinical trials using vsv-ebov vaccination. 27 recently, it has been shown that low-dose vaccination with vsv-ebov does not compromise protective efficacy in nonhuman primates. 41 lower-dose vaccination would also have a beneficial effect on vaccine manufacturing. currently, h5 hpai viruses have been classified into several clades based on phylogenetic analysis of their ha genes. 42 notably, mainly clade 2 viruses have evolved rapidly and extensively in recent years, and the continued evolution of this particular virus has heightened the concern for a pandemic. 43 thus, here we selected eight viruses from clade 2 and one virus from clade 1 (supplementary table 1 ) to investigate the crossneutralizing nature of the vaccine-induced antibody response by hi test. we found that a prime/boost vaccination with the vsvs expressing hafl elicited cross-neutralizing antibodies against all tested h5 viruses (table 1) suggesting that these vaccine vectors will likely cross-protect. the presence of hi antibodies with titers of 1:40 is considered protective 44 as demonstrated in previous animal studies using poxvirus-based vaccination. 45 crossprotection could indeed be demonstrated in mouse challenge experiments utilizing three different h5 clade 2 isolates (fig. 5) highlighting the cross-protective potential of the vaccines. while prime/boost vaccination with vsv-shazip-ebov or vsv-sha-ebov induced cross-neutralizing antibodies, the responses were generally lower in titer and detected in fewer animals. previous studies have shown that the influenza virus ha stem has the potential to induce broad protective immunity 46 and that the removal of the transmembrane domain may affect the native conformation of the ha stem potentially destroying those conformational antibody epitopes. 47 thus, the finding that our vsv vectors expressing sha/shazip did perform worse than those expressing hafl is likely due to the specific design of expressing a soluble antigen that lacks both the transmembrane and cytoplasmic domains. our studies demonstrate that vsv-based vaccines expressing hafl are superior over those expressing modified sha. however, this study did not provide any data supporting an advantage of including vsv-ebov as part of the vector design over just expressing vsv-hafl as both vectors performed similarly well with no statistically significant difference in efficacy following single-dose or prime/boost administration (fig. 2 ) nor in antibody responses (fig. 3, table 1 ). the lack of differences in ha-specific antibody responses is not necessarily in line with higher expression of hafl following vsv-hafl infection in tissue culture (fig. 1b) , but replication may be different in vivo. on the other hand, the postulated favorable immune cell targeting through vsv-ebov 30,48-50 may balance the advantage of higher antigen expression by vsv-hafl. vsv-ebov has been shown to induce rapid protective immune responses in preclinical and clinical studies. 27, 28 thus, this platform has the potential to be utilized as an emergency vaccine. while we could not demonstrate a significant difference between the vsv-hafl and vsv-hafl-ebov vaccine in regard to fast-acting properties, protection after immunization on day −1 is marginally better with vsv-ebov-hafl than vsv-hafl (fig. 4) . this difference could be due to the favorable immune cell targeting of the ebov gp, 30,48-50 but further studies with bigger animal group sizes are needed to prove this hypothesis. previous studies demonstrated that vsv-based vaccines provide rapid protection via involvement of the innate immune system combined with an early adaptive response, 26 suggesting that the vsv-ebov-hafl and vsv-hafl vaccines may induce innate immune responses that are able to control the challenge virus, allow for the adaptive immune system to catch up and lead to protection of the mice. nevertheless, the fast-acting feature makes this vaccine extremely valuable for the public health response during an epidemic or pandemic as the vaccine could be strategically administered to more vulnerable populations such as elderly and hospitalized people keeping in mind the replicative nature of the vaccine vectors. vsv-based h5 influenza virus vaccine candidates have advantages compared to the currently used influenza virus vaccines including the ease of generation of the vectors as well as the vaccine production in cell lines which are already approved for manufacturing of human vaccines. 51 a switch to cell line production would eliminate concerns regarding allergies to egg proteins. the downside of an attenuated, replication-competent vaccine approach such as vsv is adverse reactions to vaccination. however, previous preclinical vaccine work using the vsv platform, including immunization of several immunecompromised animal species, as well as clinical trials with vsv-ebov demonstrated low levels of vaccine-related adverse effects resulting in the general conclusion that the vsv vaccine platform is safe. 27, 28 in addition, vsv-based replicating vaccines are efficacious at lower doses compared to non-replicating approaches and do not require adjuvants. 52 in conclusion, we have developed two vsv-based vaccine candidates, vsv-ebov-hafl and vsv-hafl, that provide proof-ofconcept for rapid protection against hpai virus infection that are mediating cross-neutralizing responses. if clinical development confirms the promise of being fast-acting and strongly protective, vsv-based vectors might be a promising approach for the development of a pan-h5 influenza virus emergency vaccine. all infectious work was performed at the required containment level at the integrated research facility, rocky mountain laboratories (rml), division of intramural research (dir), national institute of allergy and infectious disease (niaid), national institutes of health (nih). vaccinations were carried out in bsl2 settings; h5n1s were handled exclusively under maximum containment. the animal work was approved by the institutional animal care and use committee (iacuc) and performed according to the guidelines of the association for assessment and accreditation of laboratory animal care, international and the office of laboratory animal welfare. all procedures on animals were carried out by trained and certified personnel following standard operating procedures (sops) approved by the institutional biosafety committee (ibc). humane endpoint criteria in compliance with iacuc-approved scoring parameters were used to determine when animals should be humanely euthanized. african green monkey kidney (vero e6) cells were grown in dulbecco's modified eagle's medium (dmem) (sigma-aldrich) containing 2% or 10% fetal bovine serum (fbs), 2 mm l-glutamine, 50 u/ml penicillin, and 50 μg/ ml streptomycin (all from thermo fisher scientific). baby hamster kidney (bhk)-t7 cells were grown in minimum essential medium (mem) (thermo fisher scientific) containing 10% tryptose phosphate broth (thermo fisher scientific), 5% fbs, l-glutamine, penicillin, and streptomycin. madin-darby canine kidney (mdck) cells were grown in eagle's minimum essential medium (emem) containing 10% fbs, l-glutamine, penicillin, streptomycin, mem non-essential amino acid (thermo fisher scientific), and bicarbonate (thermo fisher scientific). the h5n1 challenge viruses a/ the h5 hafl orf was constructed using the entire ha cdna sequence of h5n1 a/vietnam/1203/2004. the soluble ha (sha) orf was generated from the hafl orf by deleting the transmembrane domain and replacing the sequences encoding the polybasic cleavage site between ha1 and ha2 (pqrerrrkkrg) by one preventing cleavage (pqietrg). the sha with leucine zipper (shazip) was constructed of the sha orf by adding a gcn4pll sequence for trimerization. 35 all orfs were cloned into the patx-vsv-ebov plasmid encoding the ebov-mayinga gp. 53 replicationcompetent recombinant vsvs (vsv-ebov-shazip, vsv-shazip-ebov, vsv-sha-ebov, vsv-ebov-hafl, and vsv-hafl) were generated as described previously. 24 the complete sequence of the vsv vaccines was confirmed by sanger sequencing. detailed sequence information can be obtained from the authors upon request. the titer of each virus stock was quantified using standard plaque and tcid 50 assays on vero e6 cells. the same vaccine virus stock was used for all in vitro and in vivo work. vero e6 cells were grown to confluency in a 12-well plate and infected in triplicate with vsvwt, vsv-ebov, vsv-ebov-shazip, vsv-shazip-ebov, vsv-sha-ebov, vsv-ebov-hafl, and vsv-hafl (moi of 0.01). the inoculum was removed, cells were washed three times with dmem, and covered with dmem containing 2% fbs, 2.5 μg/ml tpck trypsin (thermo fisher scientific), and 1 u/mg na from vibrio cholerae (sigma-aldrich). tpck trypsin and na are required for the propagation of the vsv-hafl vaccine. supernatant samples were collected at 0, 24, 48, and 72 h post-infection and stored at −80°c. the titer of the supernatant samples was determined performing tcid 50 assay on vero e6 cells. samples were generated in parallel from each vaccine virus stocks produced in vero e6 cells mixed 1:1 with sodium dodecyl sulfatepolyacrylamide (sds) gel electrophoresis sample buffer containing 20% β-mercaptoethanol and heated to 99°c for 10 min. sds-page with all samples was performed in parallel on tgx criterion pre-cast gels (bio-rad laboratories) (supplementary fig. 1 ). subsequently, proteins were transferred to a trans-blot polyvinylidene difluoride membrane (bio-rad laboratories). the membrane was blocked for 3 h at room temperature in pbs with 3% powdered milk and 0.05% tween 20 (thermo fisher scientific). protein detection was performed using the following rabbit or mouse primary antibodies: anti-ha 1:1000 (cat. #11062-t54-100, sino biological inc.), anti-ebov gp (zgp 12/1.1, 1 μg/ml; kindly provided by ayato takada, hokkaido university, sapporo, japan), and anti-vsv m (23h12, 1:1000; kerafast inc.). after horse-raddish peroxidase (hrp)-labeled secondary antibody staining using either anti-mouse igg (1:10,000) or antirabbit igg (1:5000) (mouse cat. #715-035-151; rabbit cat. #711-035-152; both jackson immunoresearch), the blots were imaged using the supersignal west pico chemiluminescent substrate (thermo fisher scientific) and a fluorchem e system (proteinsimple). groups of female balb/c mice (n = 16) were vaccinated im with 1 × 10 4 pfu of the vsv-based vectors in 0.1 ml (two sites, 0.05 ml each) on day −42 and −21 (prime/boost vaccination) or −21 only (single-dose vaccination). on the day of challenge (day 0), four animals in each group were euthanized for serum collection. the remaining 12 animals in each group were challenged intranasally (in) with 100 ld 50 (400 tcid 50 ) of hpai h5n1 virus a/vietnam/1203/2004. on day 6 post challenge, four animals in each group were euthanized and samples were collected for serology. the remaining eight mice were monitored until 28 days post challenge when a terminal blood sample was collected prior to euthanasia. for the time to immunity study, groups (n = 8) of female balb/c mice were im vaccinated on day −7, −3, or −1 with 1 × 10 4 pfu of the vsv-hafl or vsv-ebov-hafl vaccine in 0.1 ml (two sites, 0.05 ml each). vsv-ebov was used as a control. all the groups were challenged in with 100 ld 50 (400 tcid 50 ) of hpai h5n1 virus a/vietnam/1203/2004. surviving mice were monitored until day 28 post infection. for the h5 cross-protection study, groups (n = 6) of female balb/c mice were im vaccinated on day −28 with 1 × 10 4 pfu of the vsv-hafl or vsv-ebov-hafl vaccine in 0.1 ml (two sites, 0.05 ml each). vsv-ebov was used as a control. all the groups were challenged in with 400 tcid 50 serum samples from h5n1-infectd mice were inactivated by gammairradiation and used in bsl2 according to ibc-approved sops. elisa plates were coated with 1 µg/ml (50 µl/well) of recombinant influenza ha (h5) (a/vietnam/1203/2004) antigen (ibt bioservices). after three washes with pbs/tween, plates were blocked with 1% bsa in pbs for 3 h at room temperature, followed by three additional washes with pbs/tween. the plates were incubated with fourfold serial dilutions of the mouse serum samples for 1 h at 37°c, and washed three times with pbs/tween. bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-mouse igg (h+l) (jackson immunoresearch) at a 1:1000 dilution and tmb substrate (kpl). the reaction was measured using the synergy™ htx multi-mode microplate reader (biotek). titers were calculated by a 4parameter curve fitting model using microsoft excel software. the cutoff value was set as the mean optical density plus three standard deviations of the control samples. hi assay hi assays were performed using eight hemagglutination units/25 μl of the different h5 viruses incubated with 25 μl of the fourfold serial dilutions of each mouse serum sample (day 0) in round-bottom 96-well plates for 1 h at room temperature. then 50 μl of a 0.8% turkey red blood cell solution (innovative research) was added to each well. plates were covered and incubated for 30 min on ice. hemagglutination titers were determined by the reciprocal of the last dilution containing agglutinated turkey red blood cells. hi titers represent the highest serum dilution that completely inhibited hemagglutination. statistical analysis was performed in prism 7 (graphpad). data presented in figs 1c, 2 (upper panels), 4 (upper panels), 5 (left panels), supplementary fig. 1c , and supplementary fig. 1d (upper panels) were examined using two-way anova with tukey's multiple comparison to evaluate statistical significance at all timepoints between all groups. significant differences in the survival curves shown in figs. 2 (lower panels), 4 (lower panels), 5 (right panels), and supplementary fig. 1d (lower panels) were determined performing log-rank analysis. data presented in fig. 3 and supplementary fig. 2 were analyzed for statistical significance using one-way anova with multiple 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protects chickens against highly pathogenic avian influenza virus h5n1 recombinant trimeric ha protein immunogenicity of h5n1 avian influenza viruses and their combined use with inactivated or adenovirus vaccines vesicular stomatitis virus vectors expressing avian influenza h5 ha induce cross-neutralizing antibodies and long-term protection poultry and the influenza h5n1 outbreak in hong kong, 1997: abridged chronology and virus isolation h5n1 highly pathogenic avian influenza: timeline of major events brief literature review for the who global influenza research agenda-highly pathogenic avian influenza h5n1 risk in humans potent vesicular stomatitis virus-based avian influenza vaccines provide long-term sterilizing immunity against heterologous challenge single low-dose vsv-ebov vaccination protects cynomolgus macaques from lethal ebola challenge toward a unified nomenclature system for highly pathogenic avian influenza virus (h5n1) antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness haemagglutination-inhibiting antibody to influenza virus cross-clade immunity in cats vaccinated with a canarypoxvectored avian influenza vaccine towards a universal influenza vaccine: different approaches for one goal enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates ebola haemorrhagic fever vsvdeltag/ebov gp-induced innate protection enhances natural killer cell activity to increase survival in a lethal mouse adapted ebola virus infection cell culture-based influenza vaccines: a necessary and indispensable investment for the future ebola vaccines in clinical trial: the promising candidates properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses we thank the animal care staff of the rocky mountain veterinary branch (niaid, nih) for their support of the animal experiments. we also thank david wentworth, vivien dugan, todd davis, and bin zhou of the virology surveillance and diagnosis branch, influenza division, centers for disease control and prevention for providing the candidate vaccine viruses and the h5n1 viruses utilized in this study. this work was funded by the division of intramural research, niaid, nih. further information on research design is available in the nature research reporting summary linked to this article. the data supporting the findings of this study are available from the corresponding author upon reasonable request. h.f. claims intellectual property regarding the vesicular stomatitis virus-based vaccines for viral hemorrhagic fevers. no other competing interests are to be disclosed. supplementary information is available for this paper at https://doi.org/10.1038/ s41541-019-0155-z.correspondence and requests for materials should be addressed to a.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. this is a u.s. government work and not under copyright protection in the u.s.; foreign copyright protection may apply 2020 key: cord-272051-arz8r204 authors: federico, maurizio title: hiv-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step date: 2011-08-15 journal: virology doi: 10.1016/j.virol.2011.05.002 sha: doc_id: 272051 cord_uid: arz8r204 the inhibitors of hiv-1 protease (pis) have been designed to block the activity of the viral aspartyl-protease. however, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. since the replication of many virus species requires the activity of host cell proteases, investigating the effects of pis on the life cycle of viruses other than hiv would be of interest. here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. these are unrelated enveloped rna viruses infecting target cells upon endocytosis and intracellular fusion. the pi-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. these findings would open the way towards the therapeutic use of pis against enveloped rna viruses other than hiv. hiv protease inhibitors (pis) are a family of small molecules specifically designed for blocking the activity of the hiv aspartylprotease (for a review, see flexner, 1998) . the pi treatment leads to the block of hiv maturation with consequent release of noninfectious viral particles (kaplan et al., 1993) . pis exert their inhibitory effect by disabling the enzyme before it can cleave the gag-pol polyprotein into its essential products. pis, together with anti-hiv compounds targeting alternative steps of the virus life cycle, are part of the most advanced anti-hiv therapies allowing millions of infected people to co-exist with the virus experiencing a good quality of life. however, it is now clear that pis can also directly or indirectly inhibit the activity of many cell proteases. in particular, pis can inhibit the activity of both 20s (andré et al., 1998) and 26s (pajonk et al., 2002) proteasome subunits, as well as that of caspases (badley, 2005) , with alterations in the susceptibility to apoptosis stimuli. both expression and release of matrixmetalloproteinases (mmps) can be also targeted by pis (bourlier et al., 2005; de barros et al., 2007) with consequences in the extracellular matrix modeling and, more in general, in the cell-cell communication. in addition, pis have been found inhibiting cell signaling pathway involving both nf-κb (dewan et al., 2009 ) and akt (kumar et al., 2009; srirangam et al., 2006) . from a clinical point of view, the pi treatment may induce beneficial effects other than those against hiv, as in the case of kaposi sarcoma regression (sgadari et al., 2002) , but can also have detrimental consequences, as for the dyslipidemia occurring in pi-treated hiv patients (calza et al., 2004) . to enter target cells, enveloped rna viruses fuse their envelope either at the cell surface in a ph-independent way, or upon internalization in intracellular vesicles through a ph-dependent mechanism (steven and spear, 2006) . in this latter case, low ph is required to induce the fusion between viral envelope and endosomal membranes, a phenomenon leading to the release of virion contents into cytoplasm. although the viral fusion process is guided by the presence of viral envelope proteins, the contribution of cell proteins is critical. using rna interference as a tool for gene expression inhibition, it was demonstrated that several cell proteins are involved in the mechanism of entry of many viruses including hiv-1 nguyen et al., 2006) , influenza (hao et al., 2008) , west nile (krishnan et al., 2008) , borna disease (clemente et al., 2010) , human hepatitis c (ng et al., 2007) , and vesicular stomatitis (vsv) (pelkmans et al., 2005) viruses. ideally, these cell products might represent potential antiviral therapeutic targets. here, the pi-induced inhibition of replication of both vsv and influenza virus is described for the first time. this was most likely a consequence of a pi-dependent block of the viral envelope fusion at the endosomes. considering that pis are well tolerated drugs in vivo, and that many relevant human pathogens belong to the family of rna viruses infecting cells through an endocytic pathway, this finding would open the way towards a broader therapeutic use of pis. hiv virions emerging from cells treated with pis remain immature viral particles as a consequence of the block of gag polyprotein cleavage. hence, pis do not affect the amounts of hiv release from infected cells, but dramatically decrease their infectivity. surprisingly enough, however, the hiv-1 release from cells infected with hiv-1 pseudotyped with the envelope protein from vsv (vsv-g) has been found inhibited by pis in single cycle replication assays. in fact, when cem gfp cells, i.e., a human t cd4 + lymphoblastoid cell line expressing gfp under the control of hiv-1 ltrs (gervaix et al., 1997) , were infected with 100 ng hiv-1 cap24 equivalent/10 5 cells of (vsv-g) δenv hiv-1, decreased percentages of hiv-1 expressing cells were observed in ritonavir-treated cells as compared to control conditions (fig. 1a) . the outcome did not change by challenging the cells with doses up to 300 ng hiv-1 cap24 equivalent/10 5 cells (not shown). conversely, and as expected, the pi-treatment had no apparent effects on single cycle replication of non-pseudotyped hiv-1, as tested in pi-treated cultures of cem gfp cells infected with wt hiv-1 (fig. 1b) . in these experiments, the presence of t-20 (i.e., a potent inhibitor of the hiv-1 env-mediated fusion) (kilby et al., 1998) added 16 h after challenge ensured that the wt hiv-1 replication was limited to a single cycle. as anticipated, ritonavir blocked the spread of wt hiv-1 in multiple-cycle replication assay (fig. 1b) . to exclude that the observed effect was a consequence of the inhibition of some yet unidentified function of the hiv-1 protease at an early step of the virus life cycle, the assay was reproduced using a vsv-g pseudotyped, pi-resistant hiv-1 strain (here referred to as pm4). this viral mutant expresses a pi-resistant viral protease as a consequence of the four amino acid substitutions in the protease gene, i.e., m 46 i , l 63 p , v 82 t , and i 84 v (condra et al., 1995) . first, the real resistance to pis of pm4 hiv-1 was checked by evaluating the percentages of cem gfp infected cells 6 days after the infection in the presence or not of ritonavir (fig. 1c) . then, cem gfp cells treated with t-20 were infected with (vsv-g) pm4 hiv-1 in the presence or not of ritonavir. this experimental setting was representative of a singlecycle replication assay since t-20 disabled both hiv-1 env-mediated viral entry and possible cell re-infections. similarly to what observed with (vsv-g) δenv hiv-1 strain, a significant inhibitory effect of ritonavir on the hiv-1 expression was detected in (vsv-g) pm4 challenged cells (fig. 1d ). this strongly suggests that the viral protease activity was not involved in the pi-induced inhibition of (vsv-g) hiv-1 expression. overall, these results can be a consequence of a still unrecognized inhibitory effect of pis on some step of the viral entry driven by vsv-g. next, the possibility that the pi inhibitory effect was operative also against vsv was investigated. to this end, hela cells were pre-treated overnight with different concentrations of six pis, i.e., ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir. then, the cells were challenged with 0.2 m.o.i. of vsv. after 1 h of adsorption in a small volume, the viral inoculum was removed, the cells extensively washed, and incubated for additional 8 h in the presence of pis. afterwards, the supernatants were harvested and titrated for the amounts of infectious vsv. as depicted in figs. 2a-b, a strong reduction of vsv replication (more than 2 logs) was observed when the cells were treated with 2.5 μm saquinavir and nelfinavir. the inhibitory effect appeared more potent (up to about 4 logs) by increasing the concentration of these pis to 10 μm, and remained significant (i.e., more than 1 log) by increasing the m.o.i. up until 1 (not shown). no differences were observed when the pis pretreatment was carried out for 2, 4, 6, or 8 h (not shown). as calculated through a wide dose-response curve, the 50% inhibitory concentration (ic 50 ) values of saquinavir and nelfinavir were 1.4 and 1.1 μm, respectively. on the other hand, significant inhibition of viral replication were detected in ritonavir and indinavir treated cells starting to the concentrations of 20-30 μm (fig. 2b) . the ic 50 of ritonavir and saquinavir were 16 and 14 μm, respectively. the idea that pis negatively affect the vsv replication was further strengthened by performing a set of more stringent challenge experiments. in detail, hela cells were pre-treated with optimal concentrations of the most effective pis, and then infected with 2-fold decreasing amounts of vsv starting to m.o.i. 2. the cytopathic effect was monitored 24 h post challenge. the outcome of this assay was scored in terms of the viral dilutions no more able to induce cytopathic effect. notably, in these conditions an about 30-fold reduction of vsv infectivity was observed in cells treated with saquinavir or nelfinavir (fig. 2c ). since the average replication time of vsv is 6-8 h (whelan et al., 2004) , in this experimental setting vsv was expected to complete at least three replication cycles in control cells. then, it was controlled whether the antiviral effect was a consequence of a general cytotoxicity of pis. to this aim, cells were treated with different pi concentrations for 8 h, then labeled with carboxyfluorescein diacetate succinimidyl ester (cfse), and thereafter refed with complete medium in the presence of pis. cfse was expected to be equally distributed upon cell division, then resulting in a halving of the overall cell fluorescence after cell duplication. cytotoxic effects were measured in terms of inhibition of the cell duplication as detectable by the impairment of the halving of the cfse-associated cell fluorescence. fig. 2d reports the mean fluorescence intensities (mfis) measured in cell cultures treated with different concentrations of the pis active against vsv replication. block of cell duplication with cell mortality over 20% has been detected using 100 μm ritonavir, saquinavir, and nelfinavir, while indinavir showed a much lower cytotoxic effect. the 50% cytotoxic concentration (cc 50 ) values of pis ( fig. 2d , insert) were calculated as the doses inhibiting by 50% the fluorescence halving, expectedly a consequence of the block of the duplication in 50% of cells. the therapeutic indexes (i.e., cc 50 divided by ic 50 ) appeared higher than 22 for both saquinavir and nelfinavir. this is suggestive of specific antiviral effects for these pis. taken together, these data reveal a previously unrecognized effect of pis against vsv. in addition, these results support the idea that the pi-induced reduction of (vsv-g) hiv-1 expression is a consequence of the inhibition on some step of the viral entry involving the vsv-g envelope protein. to add relevance to our findings, the possibility that the inhibitory effect of pis applied also to alternative virus species infecting by phdependent fusion was investigated. to this aim, the well characterized influenza virus/mdck cell system was considered. mdck cells were treated overnight with different pis, and then challenged with m.o.i. 0.1 and 0.5 of the pr8 influenza virus. after challenge, the cells were extensively washed, and reseeded in medium without serum in the presence of both pis and tosylamido-2-phenil ethyl chloromethyl ketone (tpck)-treated trypsin. twenty-four h later (i.e., the expected time for the completion of the replication cycle of influenza virus) (sidorenko and reichl, 2004; smith and ribeiro, 2010) , the supernatants were harvested and titrated for the contents of infectious particles. a strong inhibition of the replication of influenza virus (up to 4 logs at the lower m.o.i.) has been detected with 10 μm saquinavir and 5 μm nelfinavir (fig. 3a) . ritonavir inhibited the viral replication less efficiently (fig. 3a) , while indinavir, amprenavir and atazanavir appeared ineffective (not shown). in the interpretation of these data, it should be emphasized that the inhibitory effect of pis cannot be a consequence of a pi-induced impairment of the activation of the hemagglutinin envelope protein (ha) of the influenza virus, which is a step required for productive infection (steinhauer, 1999) . in fact, influenza virus preparations used for challenges were exclusively recovered from the allantoic fluid of embryonated chicken eggs, where ha is cleaved by egg proteases. consistently, tpck-trypsin did not influence the infectivity of the virus preparations in single cycle replication assay (not shown). the cell cytotoxicity of pis active against influenza virus was then evaluated in mdck cells similarly to what above described for hela cells (fig. 3b ). block of cell duplication in the presence of cell mortality over 20% has been detected with 100 μm ritonavir and saquinavir, and with 50 μm nelfinavir. the therapeutic indexes appeared higher than 15 for both saquinavir and nelfinavir. the assay gave similar results when the cells were analyzed 24 h after cfse labeling (not shown). taken together, these data suggest that pis could target the replication of diverse enveloped viruses infecting by a ph-dependent endocytic pathway. next, it was investigated whether the pis most effective against vsv and influenza virus replication counteract also the replication of viruses infecting by a ph-independent mechanism. as a proof of principle, the pis were assayed against the replication of newcastle disease virus (ndv), i.e., a paramixovirus infecting upon fusion at the cell surface. mdck cells were pre-treated with 2.5 to 10 μm saquinavir or nelfinavir, and then infected with 2-fold decreasing amounts of ndv starting to m.o.i. 1. the cytopathic effect was monitored 48 h post challenge. no significant reductions in the ndv infectivity have been noticed in pi-treated as compared to control cell cultures (fig. 4) . these results indicate that pis do not affect the replication of viruses entering by a ph-independent way. the inhibitory effect is operative when pis are co-administered with the infecting virus to identify the virus replication step targeted by pis, the effects of pis added before and/or after viral challenge were evaluated. the assays were carried out using both hela/vsv and mdck/influenza virus systems. in a first set of experiments, the pi treatment was discontinued just before the virus challenge. then, the cells were infected with vsv or influenza virus at m.o.i. of 0.2 and 0.5, respectively. after 1 h of adsorption, the cells were extensively washed and refed in the appropriate medium. in these conditions, no inhibition of the replication of both vsv and influenza virus was detectable (fig. 5a) . alternatively, pis were added soon after the virus adsorption or at different times after the challenge within a timeframe of 8 h (for vsv infection) and 24 h (in the case of challenge with influenza virus), i.e. the respective replication times. the supernatants were harvested 8 h (for vsv) and 24 h (for influenza virus) post-infection, and titrated for the amounts of infectious virus particles. it appeared that pis inhibited the virus replication also when added early after challenge (fig. 5b) . the inhibitory effect dropped shortly in the case of vsv infection, and more gradually when cells were infected with influenza virus. saquinavir and nelfinavir have shown similar inhibitory efficiencies (not shown). these results indicate that the presence of pis at early times after virus challenge is mandatory for the inhibitory effect, consistently with the idea that pis target an early event of the replication cycle of both viruses. vsv, and (vsv-g) hiv-1 in an hiv-1 protease independent manner, further supported the idea that pis would influence some common early replication step, e.g., virus attachment, endocytosis, and/or endosomal fusion. to dissect among these different possibilities, the intracellular fate of challenging virus was followed using gfp-labeled hiv-1-based vlps (muratori et al., 2006) pseudotyped with either wt or fusiondefective vsv-g (fig. 6a) . the fluorescence of these vlps relies on the high incorporation levels of the product of fusion between gfp and a hiv-1 nef mutant acquiring a palmitoylation site at its n-terminus as the consequence of the g to c substitution at the amino acid 3. first, the possible effects of pis on the virus attachment on target cells were investigated. to this end, 10 5 hela cells treated or not with pis were challenged with 500 ng of (wt vsv-g) nef g3c -gfp vlps for 1 h at 4°c. afterwards, the cells were extensively washed and facs analyzed. to control that the cell-associated fluorescence was not a consequence of endocytosed vlps, a part of the cell samples was treated with trypsin. it appeared that the treatment with pis did not significantly affect the cell binding of fluorescent vlps (fig. 6b ). this strongly suggests that pis do not interfere with the binding of viral particles on the cell membrane. next, the influence of pis on virus endocytosis was investigated. to this aim, gfp-fluorescent vlps incorporating a vsv-g mutant (here referred to as fd vsv-g) unable to support the ph-dependent fusion were used. the impaired fusion activity was a consequence of the a to k amino acid substitution at the position 133 (fredericksen and whitt, 1995) . the use of this envelope protein mutant was associated with the trypsin treatment just before the facs analysis to ensure that the cell-associated fluorescent signal exclusively referred to the intracellular accumulation of vlps. hela cells treated or not with pis were challenged with 500 ng hiv-1 cap24 equivalents of (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and incubated for 3 h at 37°c in the presence or not of pis. then, the cells were treated with trypsin, and the cell-associated gfp fluorescence was evaluated by facs analysis. as control, vlp-challenged cells were incubated at 4°c before the trypsin treatment. again, no apparent differences in the cellassociated fluorescence were detected between control and pi-treated cells (fig. 6c) , suggesting that pis have no influence on the efficiency of the endocytosis of viral particles. this conclusion was enforced by the results obtained through an alternative experimental approach where control or pi-treated hela cells were infected with 0.2 pfu/cell of vsv. thirty and 60 min later, the cells were extensively washed, treated with trypsin to leave out non-adsorbed viral particles, lysed, and analyzed by western blot for the presence of vsv-g. no significant reduction of the vsv-g specific signals was detectable at both time points in pi-treated cells as compared to control conditions (fig. 6d) . considering that the neosynthesis of vsv-g starts at least 90 min post-infection (rothman and lodish, 1977) , these results, consistently with the data obtained with fluorescent vlps, suggest that pis do not affect the endocytosis of viral particles. the results from these experiments indicate that the inhibitory effect of pis is not the consequence of impaired attachment and endocytosis of viral particles. next, the possibility that the inhibitory effect of pis was a consequence of a defect in the endosome to cytoplasm virus delivery was investigated. to this end, the differences in the cell-associated fluorescence levels in cells internalizing wt or fd vsv-g pseudotyped nef g3c -gfp vlps were exploited. in fact, using the same experimental conditions described for the endocytosis assay, it was reproducibly observed that, early after vlp challenge, the mean fluorescence intensity (mfi) of cells challenged with (wt vsv-g) vlps was near 2-fold higher than that of cells treated with (fd vsv-g) vlps. more significantly, in the latter condition both percentages of fluorescent cells and mfi heavily dropped 16 h after challenge. on the contrary, both facs parameters appeared only slightly reduced in cells challenged with (wt vsv-g) vlps (fig. 7a ). these differences can be explained by the fact that, due to the inability of fd vsv-g to induce fusion and delivery of the vlp contents into cytoplasm, the nef g3c -gfp molecules incorporated in (fd vsv-g) vlps were addressed to rapid degradation into the endosomal/lysosomal compartment. conversely, the efficient fusion between viral envelope and endosome membranes induced by wt vsv-g allowed the release of nef g3c -gfp molecules into cytoplasm, where they are expected to be degraded with a kinetic much slower than that operating in endosomes/lysosomes. next, pi-treated or untreated hela cells were challenged with 500 ng hiv-1 cap24 equivalent of either (wt vsv-g) or (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and 3 and 16 h later, the cells were treated with trypsin. facs analysis of the cell-associated gfp fluorescence revealed that the pi treatment did not affect the fluorescence levels in cells challenged with (fd vsv-g) nef g3c -gfp vlps at both time points. on the contrary, after 3 h it was observed a relevant reduction in the mfi within pi-treated cells challenged with (wt vsv-g) nef g3c -gfp vlps as compared with untreated cells. more strikingly, a strong decrease of both mfi and percentage of fluorescent cells was detectable in pi-treated cells at 16 h post-challenge (fig. 7b) . as expected, among the different pi tested, saquinavir and nelfinavir produced the strongest inhibitory effect (data not shown). next, dose-response endocytosis assays using different concentrations of nelfinavir were carried out by analyzing the cell-associated fluorescence 16 h after the challenge. the results showed that, within the cells challenged with (wt vsv-g) nef g3c -gfp vlps, the pi treatment reduced both mfi and percentages of fluorescent cells in a dose-dependent manner. at the highest pi concentrations, both parameters reached levels similar to those detectable in cells challenged with (fd-vsv-g) nef g3c -gfp vlps (fig. 7c) . hence, it seemed that the pi treatment diverted the fate of endocytosed (wt vsv-g) towards that of (fd vsv-g) vlps. this strongly suggests that pis negatively affect the fusion of viral envelope with the endosomal membranes. together, these results support the idea that the inhibitory effect of pis acts at the level of the delivery of the viral particles from endosomes to cytoplasm of infected cells. a possible explanation for the apparent block of viral envelope fusion could be that pis may increase the ph in endosomes in a way to inhibit the low ph-dependent conformational changes needed for both vsv-g and influenza ha to switch the fusion process. to test whether pis affect intracellular ph, both hela and mdck cells were labeled with lysosensor green dnd-189. this reagent becomes fluorescent only in acidic intracellular compartments, meanwhile exhibiting decreased fluorescence intensity upon ph increase. thus, it represents a useful reagent for detecting possible ph variations in endosomes. cells were treated overnight with the pi doses most effective against vsv and/or influenza virus replication, then labeled with lysosensor green dnd-189 for 30 min in the presence of pis, and finally analyzed by facs. as control, cells treated with bafilomycin a1, i.e., a powerful inhibitor of the vacuolar atpase proton pump, were also tested. as depicted in fig. 8 , no significant variations in the cellassociated fluorescence have been detected in pi-treated cells as compared with control conditions, indicating that pis do not affect the intracellular ph. these results strongly suggest that the pi-induced block of virus delivery in cytoplasm would not be a consequence of the increase of endosomal ph. viruses hijack cell functions to replicate in host cells. hence, the selective targeting of cell products supporting virus replication could represent a successful antiviral strategy. the results from the here described investigations indicate that drugs designed to counteract the activity of the hiv protease show a strong inhibitory effect against activity(ies) of target cells. the antiviral potency of the most effective pis reached about 4 logs in the reduction of virus yields, with therapeutic indexes higher than 15. the pi-induced effect against vsv appeared comparable in magnitude to that observed in cell treated with type-1 interferons (masters and samuel, 1983) . on the other hand, the potency of pis against the influenza virus replication appeared comparable to that of most potent inhibitors. among these, t-705 (furuta et al., 2002) reduced the viral yield of the most susceptible influenza strain tested of at best 2.7 logs at the concentration of 5 μm upon mdck cell challenge with 0.01 m.o.i. (sleeman et al., 2010) . das181, i.e. a sialidase fusion protein, blocked the influenza virus replication at sub-micromolar concentrations, however when mdck cells were challenged with 0.001-0.005 m.o.i. (triana-baltzer et al., 2009) . conversely, micromolar concentrations of pis induced more than 3 logs of reduction of the influenza virus yield from mdck cells challenged with 0.5 m.o.i. in the challenge experiments where pis were added soon after the adsorption of vsv or influenza virus inocula, the antiviral effect appeared slightly reduced as compared with control conditions where pis were maintained throughout. considering also the results obtained in the endocytosis assays, it is conceivable that this subtle difference could be a consequence of the accession to cytoplasm of a small amount of viral particles during the adsorption time and before the pi treatment. the possibility that pis affect some virion structural component in a way to hinder the process of fusion in endosomes appeared unlikely. in fact, the treatment of 10-fold concentrated vsv or influenza virus preparations with up to 500 μm pis for 30 min before challenge did not produce significant decrease of the viral yields (not shown). however, we cannot formally exclude that pis inhibit some yet unidentified protease activity associated with vsv-g or influenza virions. in this regard, it has been proposed that pis could negatively affect the chymotrypsin-like protease activity of the pa subunit of the rna-directed rna polymerase of influenza virus (savarino, 2005) . furthermore, it was reported that nelfinavir reduces the replication of sars coronavirus (yamamoto et al., 2004) likely as a consequence of the inhibition of the 3c-like viral protease (for a review, see 35). the results from here reported experiments indicate that pis act on early events of viral replication, but not on virus attachment and endocytosis. this latter finding was consistent with previously reported results indicating that pis do not affect the endocytosis of hiv-1 particles in dendritic cells (muratori et al., 2009) . rather, it appeared that pis interfere with the delivery of viral particles from endosomes to cytoplasm. this did not seem to depend on a pi-induced increase of the endosomal ph possibly inhibiting the low phdependent conformational changes of viral envelope proteins required for viral fusion. the concept that the activity of cell proteases is part of the mechanisms underlying the replication of many virus species is widely accepted. for example, the cleavage of ha generated by cell proteases is required for the activation of the viral envelope protein preceding the viral fusion of influenza virus (klenk et al., 1975; lazarowitz and choppin, 1975) . similarly, the activation of envelope proteins of ebola (schornberg et al., 2006) , nipah (pager and dutch, 2005) , and corona viruses (bosch et al., 2008) is regulated by cathepsins, i.e., a family of cell aspartyl-proteases. the identification of host factors, in particular aspartyl-proteases, involved in the here described pi-induced inhibition of viral entry deserves further investigations. the results from functional genetic screens have demonstrated that alternative cell proteases act as co-factors in the replication of different viruses clemente et al., 2010; hao et al., 2008; krishnan et al., 2008; ng et al., 2007; nguyen et al., 2006; pelkmans et al., 2005) . concerning vsv, the comparison of these data with those regarding the cell proteases known to be sensitive to pis identifies both proteasome subunits as possible relevant pi targets (clemente et al., 2010 ). this appears consistent with the recently reported evidence that mg132 (i.e., a proteasome inhibitor) inhibits vsv replication (neznanov et al., 2008) . alternative functional genetic screens revealed that also mmp-21 could be part of the mechanism of vsv replication (clemente et al., 2010) . considering that the activity of mmp-21, like other mmps, might be affected by pis, it would be of interest investigating the role of this mmp in the here described antiviral effect of pis. the here reported findings would open the way towards preclinical assays designed to test the potency of pis against in vivo infections sustained by orthomyxo-and rabies viruses. it will be also of interest extending the investigations on additional pathogenic enveloped viruses infecting by endocytosis. vsv-g pseudotyped hiv-1 preparations were obtained from supernatants of 293t cells 48 h after co-transfection with a cmv immediate-early promoted vsv-g-expressing vector and vectors expressing nl4-3 hiv-1, its δenv derivative, or pm4 hiv-1 (molar ratio 1:5) performed by the lipofectamine 2000-based method (invitrogen). supernatants were clarified and concentrated by ultracentrifugation as described (federico et al., 2001) . virus preparations were titrated by measuring hiv-1 cap24 contents by quantitative enzyme-linked immunosorbent assay (elisa; innogenetic). both preparations and assays of vsv (indiana strain) have been performed basically as described (gresser et al., 1968) . briefly, high titer stocks of vsv have been prepared upon infection of hela cells with at low multiplicity of infection (m.o.i.), i.e. b0.01 plaque forming unit (pfu)/cell. supernatants were harvested 24 h later, clarified, stocked, and frozen. vsv titrations of high titer stocks have been carried out by plaque method. the a/puerto rico (pr)/8/34 h 1 n 1 human influenza virus was grown in 11-day-old embryonated chicken eggs. the allantoic fluid was clarified by centrifugation at 5000×g for 15 min at 4°c. the virus was pelleted by centrifugation at 65,000×g for 1 h at 4°c and resuspended in 1 ml of phosphatebuffered saline. portions of the solution were stored as aliquots at −20°c. influenza preparations were titrated by standard plaque assay on mdck cells. virus titers for these stocks ranged from 1 to 8 × 10 7 pfu/ml. preparations of the hertz strain of ndv were recovered after 48 h of incubation in 9-11-day-old embryonated chicken eggs inoculated in the allantoic cavity. after harvesting, the allantoic fluid was clarified by centrifugation at 10,000×g, and titrated as infectious units/ml through the end-dilution method by assessing the cytopathic effect on mdck cells 48 h post infection. the titer of the viral stock used was 2.1 × 10 7 infectious units/ml. cem gfp cells were grown in roswell park memorial institute (rpmi) medium supplemented with 10% heat-inactivated fetal calf serum (fcs). hela, mdck, 293t and 293/gpr inducible hiv-1 packaging cells (sparacio et al., 2001) were grown in dulbecco's modified eagle's medium plus 10% fcs. infections of cem gfp cells with hiv-1 or pseudotyped derivatives were carried out by spinoculation at 400 × g for 30 min at room temperature (r.t.) using 200 ng and 50 ng cap24 equivalent of hiv-1 and (vsv-g) hiv-1/10 5 cells, respectively. then, virus adsorption was prolonged for additional 2 h at 37°c and, finally, cells were washed and refed with the complete medium. hela cells were infected with vsv by adsorbing the viral inoculum for 1 h at 37°c in a small volume (e.g., 0.1 ml of serum free medium for 2 × 10 5 cells in 12 well plates). thereafter, the cells were extensively washed, and refed with appropriate complete medium. mdck cells were used as targets of pr8 influenza virus. the challenges were carried out as for vsv infection, except that after virus adsorption, cells were refed with medium without serum in the presence of 1 μg/ml of tpck-treated trypsin (worthington biochemical corporation). hela and mdck cells served to titrate infectious vsv and influenza virus in supernatants harvested after challenge experiments. the titrations were carried out by the end-dilution method with triplicate conditions by challenging the cells with 3-fold scaled supernatant dilutions (in the presence of 1 μg/ml tpck-trypsin for influenza virus titrations), and by evaluating the cytopathic effect after 36 h (for vsv) and 72 h (for influenza virus). concentrated virus preparations previously titrated by the plaque assay were used as standards. for ndv infections, mdck cells were challenged with 2-fold decreasing viral dilutions starting to m.o.i. 1, and the cythopatic effect was assessed 48 h later. t-20, ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir were obtained from the nih aids research and reference reagent program. both control and pi-treated cells were labeled with 1 μm cfse (molecular probes, invitrogen) following the manufacturer's recommendations. a cell sample was immediately processed to determine the fluorescence levels at the zero time. thereafter, the remainder cell cultures were refed with complete medium and, at the time of completion of one cell duplication (on the average, 12 h for both hela and mdck cells at their logarithmic phase), were harvested, and the fluorescence measured by facs analysis. dead cells were identified upon labeling with 5 μg/ml of propidium iodide (sigma-aldrich). preparation of fluorescent vlps, challenge, and detection assays fluorescent vlps were obtained as previously described (muratori et al., 2009) . briefly, 293/gpr hiv-1 packaging cells were cotransfected with vectors expressing the green-fluorescent protein (gfp) fused at its n-terminus with a g 3 c hiv-1 nef mutant, together with a vector expressing wt or fusion-defective (fd) vsv-g (fredericksen and whitt, 1995) . supernatants were harvested 2 days later, concentrated by ultra-centrifugation on 20% sucrose cushion, and titrated for the hiv-1 cap24 contents. for hela cell challenge, 500 ng cap24 equivalent of fluorescent (vsv-g) hiv-1 vlps/10 5 cells were adsorbed for 1 h at 4 or 37°c in a volume of 0.1 ml in 48 well plates. thereafter, 0.1 ml of complete medium was added and, finally, the cells were extensively washed and analyzed by facs. in the endocytosis assays, the facs analysis was carried out after incubation with trypsin for 15 min at 37°c. both cells and purified vlp preparations were lysed in pbs, 1% triton x-100 in the presence of anti-proteolytic agents. for the preparation of cytoplasmic extracts, whole cell lysates were centrifuged at 6000×g for 10 min at 4°c, and the supernatants frozen at −80°c. aliquots of 200 ng hiv-1 cap24 equivalent of vlps and of 30 μg of total cell proteins were separated in 10% sds-page, and then transferred by electroblotting on nitrocellulose membranes (sartorius ag) for 60 min at 100 v with a bio-rad transblot. nitrocellulose membranes were blocked in 3% bovine serum albumin (bsa) fraction v (sigma) in ttbs/edta (10 mm tris-hcl, ph 7.4; 100 mm nacl; 1 mm edta; 0.1% tween-20) for 30 min at room temperature, then incubated for 1 h at r.t. with specific antibodies diluted in 1% bsa/ ttbs-edta. the following abs served for the revelation of both vlpand cell-associated products: arp 444 sheep anti-nef antiserum from mark harris, university of leeds, leeds, uk; rabbit polyclonal anti-vsv-g abs from immunology consultant laboratories; monoclonal anti human β-actin from amersham pharmacia biotech. immune complexes were detected through horseradish peroxidase-conjugated goat anti-sheep, anti-rabbit (both from calbiochem) and antimouse abs (nen), followed by enhanced chemioluminescence reaction (euroclone). the lysosensor probe dnd-189 (molecular probes, invitrogen) served to detect possible changes in the endosomal ph upon pi treatment. this is an acidotropic probe accumulating in acidic organelles which exhibits decreasing fluorescence upon ph increase. cells pre-treated with pis or, as control, with bafilomycin a1 (sigma-aldrich) were labeled with 1 μm of lysosensor dnd-189 in complete medium for 30 min in the presence of the drugs. afterwards, cells were extensively washed, fixed, and fluorescence evaluated by facs analysis. when appropriate, data are presented as mean + standard deviation values (sd). in some instances, statistical analysis was performed according to paired student's t-test, and confirmed using the non-parametric wilcoxon rank sum test. p-values b0.05 were considered significant. an inhibitor of hiv-1 protease modulates proteasome activity, antigen presentation, and t cell responses in vitro and in vivo effects of hiv protease inhibitors on apoptosis cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide protease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiation identification of host proteins required for hiv infection through a functional genomic screen dyslipidaemia associated with antiretroviral therapy in hiv-infected patients identification of host factors involved in borna disease virus cell entry through a small interfering rna functional genetic screen in vivo emergence of hiv-1 variants resistant to multiple protease inhibitors inhibition of human preadipocyte proteasomal activity by hiv protease inhibitors or specific inhibitor lactacystin leads to a defect in adipogenesis, which involves matrix metalloproteinase-9 an hiv protease inhibitor, ritonavir targets the nuclear factor-kappab and inhibits the tumor growth and infiltration of ebvpositive lymphoblastoid b cells hiv-1 nef activates stat1 in human monocytes/macrophages through the release of soluble factors hiv-protease inhibitors vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity vitro and in vivo activities of anti-influenza virus compound t-705 a new reporter cell line to monitor hiv infection and drug susceptibility in vitro effect of repeated inoculation of interferon preparations on infection of mice with encephalomyocarditis virus drosophila rnai screen identifies host genes important for influenza virus replication partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry activation of influenza a viruses by trypsin treatment rna interference screen for human genes associated with west nile virus infection ritonavir blocks akt signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion u cells by cloned human leukocyte interferon. i. effect on early and late stages of the viral multiplication cycle generation and characterization of a stable cell population releasing fluorescent hiv-1-based virus like particles in an inducible way human immunodeficiency virus type 1 (hiv-1) protease inhibitors block cell-to-cell hiv-1 endocytosis in dendritic cells different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology unpaking" human immunodeficiency virus (hiv) replication: using small interfering rna screening to identify novel cofactors and elucidate the role of group i paks in hiv infection cathepsin l is involved in proteolytic processing of the hendra virus fusion protein the human immunodeficiency virus (hiv)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-hiv-associated human cancer cells genome-wide analysis of human kinases in clathrin-and caveolae/raftmediated endocytosis synchronised transmembrane insertion and glycosylation of a nascent membrane protein expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein hiv protease inhibitors are potent antiangiogenic molecules and promote regression of kaposi sarcoma structured model of influenza virus replication in mdck cells in vitro antiviral activity of favipiravir (t-705) against drug-resistant influenza and 2009 a(h1n1) viruses modeling the viral dynamics of influenza a virus infection generation of a flexible cell line with regulatable, high-level expression of hiv gag/pol particles capable of packaging hiv-derived vectors effects of hiv protease inhibitor ritonavir on akt-regulated cell proliferation in breast cancer role of hemagglutinin cleavage for the pathogenicity of influenza virus biochemistry. viral glycoproteins and an evolutionary conundrum novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein transcription and replication of nonsegmented negative-strand rna viruses hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus this work was supported by grants from the aids project of the ministry of health, rome, italy. t-20 and pis were obtained from the nih aids research and reference program. i thank p. borghi, department of cellular biology and neuroscience, istituto superiore di sanità, rome, italy for kindly supplying both vsv and ndv preparations. i also thank a.r. castrucci and a.m. ciccaglione, department of infectious, parasitic and immunomediated diseases, istituto superiore di sanità, rome, italy, for kindly providing influenza virus preparations and the vector expressing fd vsv-g, respectively. i'm indebted to g. fornari luswergh for her excellent editorial assistance. key: cord-296466-hakaoo9i authors: mäkelä, anna r.; oker‐blom, christian title: baculovirus display: a multifunctional technology for gene delivery and eukaryotic library development date: 2006-09-22 journal: adv virus res doi: 10.1016/s0065-3527(06)68003-2 sha: doc_id: 296466 cord_uid: hakaoo9i for over a decade, phage display has proven to be of immense value, allowing selection of a large variety of genes with novel functions from diverse libraries. however, the folding and modification requirements of complex proteins place a severe constraint on the type of protein that can be successfully displayed using this strategy, a restriction that could be resolved by similarly engineering a eukaryotic virus for display purposes. the quite recently established eukaryotic molecular biology tool, the baculovirus display vector system (bdvs), allows combination of genotype with phenotype and thereby enables presentation of eukaryotic proteins on the viral envelope or capsid. data have shown that the baculovirus, autographa californica multiple nucleopolyhedrovirus (acmnpv), is a versatile tool for eukaryotic virus display. insertion of heterologous peptides and/or proteins into the viral surface by utilizing the major envelope glycoprotein gp64, or foreign membrane‐derived counterparts, allows incorporation of the sequence of interest onto the surface of infected cells and virus particles. a number of strategies are being investigated in order to further develop the display capabilities of acmnpv and improve the complexity of a library that may be accommodated. numerous expression vectors for various approaches of surface display have already been developed. further improvement of both insertion and selection strategies toward development of a refined tool for use in the creation of useful eukaryotic libraries is, however, needed. here, the status of baculovirus display with respect to alteration of virus tropism, antigen presentation, transgene expression in mammalian cells, and development of eukaryotic libraries will be reviewed. in the era of genomics and proteomics, direct coupling of proteins to their dna-coding sequence is extremely valuable as it holds potential to derive functional information from unknown open reading frames. proteins can be identified by virtue of their unique functional properties and their encoding gene subsequently isolated. replicating nanoparticles, such as bacteriophages, have proven ideal for this type of application as they can be designed to display peptides or proteins of interest on their surface while encapsidating the gene of interest. due to the exceptional titers that phages can achieve, the diversity of the resultant prokaryote-based libraries is very high. successful examples of such a display technology include isolation of antibodies from large combinatorial libraries displayed on the surface of bacteriophages. phage display, however, has notable limitations due to the simple posttranslational machinery provided by the prokaryotic host. the eukaryote-based baculovirus expression vector system (bevs), primarily based on the use of autographa californica multiple nucleopolyhedrovirus (acmnpv), was developed during the 1980s (luckow and summers, 1988; miller, 1988a miller, ,b, 1989 sherman and mcintosh, 1979; smith et al., 1983) . complex animal, human, and viral proteins, requiring folding, subunit assembly, and/or extensive posttranslational modification, can be successfully expressed using this system (kost et al., 2 00 5) . the successful and wide adoption of bevs benefits the choice of acmnpv as a candidate for the development of a safe eukaryotic display system aimed at proper presentation of antigens, gene delivery to mammalian cells as well as development of eukaryotic libraries. surface glycoproteins of enveloped viruses are attractive candidates for control and manipulation of cellular recognition. the limitations of prokaryotic display systems regarding posttranslational modifications and folding of the displayed proteins has led to the development of alternative eukaryotic display systems. during the last decade, the expression of foreign peptides and proteins on the baculoviral surface has been quite extensively studied and display has already been employed in a number of applications. although an insect virus, the tropism and transduction efficiency of acmnpv with respect to mammalian cells or tissues can be manipulated by a variety of techniques including mutation of the major viral envelope protein (gp64), incorporation of targeting peptides or antibodies into virions, and vector pseudotyping (fig. 1) . the extensive diversity of mammalian cells that can be transduced by baculovirus vectors implies that the entry and uptake mechanisms of this insect virus by mammalian cells are universal (kost and condreay, 2002) . therefore, several strategies have been developed to restrict viral transduction to desired cell types. baculovirus transduction has generally been considered as safe and nontoxic to mammalian cells, and cell growth has not been stalled even at notably high mois (ho et al., 2004) . baculovirus enters insect cells by endocytosis followed by a low-ph-induced fusion of the viral envelope with the endosomal membrane, consequently permitting viral entrance into the cytoplasm and nucleus (dee and shuler, 1997) . correspondingly, baculovirus is considered to enter mammalian cells via the same route ( fig. 1) , as gene expression is inhibited by lysosomotropic agents that inhibit endosomal maturation (boyce and bucher, 1996; hofmann et al., 1995; van loo et al., 2001) . in addition to clathrin-mediated endocytosis, baculovirus is presumably internalized by macropinocytosis . although the receptor molecule(s) of acmnpv are unknown, the cell surface molecules for the attachment and entry of the virus have been suggested to involve common constituents of the cell membrane including phospholipids or heparan sulfate proteoglycans (duisit et al., 1999; tani et al., 2001) . despite the somewhat limited knowledge regarding the molecular mechanism involved in baculovirus entry, functional alteration of baculovirus tropism has been achieved. gp64 is the major baculoviral envelope (phospho)glycoprotein (whitford et al., 1989) that is present on the surface of infected insect cells and on budded virions as homotrimers, forming typical peplomer structures at the pole of the virion (markovic et al., 1998; oomens et al., 1995) . for the budded form of acmnpv, it has been shown that gp64 determines the viral receptor preference in inhibition studies with a monoclonal anti-gp64 antibody and, therefore, defines both the host range and the infection efficiency of the host (hohmann and faulkner, 1983; volkman and goldsmith, 1985) . gp64 is necessary for the low-ph-triggered membrane fusion activity (blissard and wenz, 1992; jarvis and garcia, 1994; markovic et al., 1998; oomens et al., 1995; plonsky et al., 1999) and is essential for viral budding from insect cells as well as spreading the infection through cell-to-cell transmission (monsma et al., 1996) . permissive epitope insertions into the gp64 have been achieved without altering or disturbing viral infectivity. one example of this approach is a study where ernst et al. (2000) took advantage of the naturally occurring noti restriction site of gp64 at amino acid position 278 and nondestructively inserted two short peptides, that is, eldkva of the human immunodeficiency virus type 1 (hiv-1) gp41 and an eight amino acid streptavidin binding streptagii into the coding region of gp64. in a subsequent study, the eldkva peptide was also inserted into 17 different positions of gp64, where viral propagation was retained in as many as 13 cases, indicating that insertions of the affinity tags did not considerably affect the expression or function of gp64 (spenger et al., 2002) . thus, small peptides with specific and high affinities to receptors on mammalian cells could be introduced into gp64 for targeting to desired cell types. while direct modification of native gp64 may be advantageous (ernst et al., 1998 (ernst et al., , 2000 spenger et al., 2002) , fusion of heterologous proteins and ligand-binding moieties to an extra copy of the gp64 gene fig 1. a generalized schematic outline of the multifunctional baculovirus display technology used for eukaryotic library development and mammalian gene delivery. entry of baculovirus into mammalian cells is thought to be similar to that for insect cells. the receptor molecule(s) for baculovirus binding and entry are unknown, but they have been suggested to involve common cell surface components. the virus enters mammalian cells by endocytosis followed by low-ph-induced membrane fusion with endosomes and capsid release into the cytoplasm. the capsid is then transported toward the nucleus along actin filaments and enters the nucleus presumably through nuclear pores followed by uncoating and release of the genome. through incorporation of ligands with high and specific affinities into the virus envelope, it is possible to target baculoviral transduction to desired cell types expressing the receptor molecule for the displayed ligand. foreign peptides or proteins can be displayed on the baculovirus envelope as n-terminal (a) or internal (b) fusions to gp64, using a heterologous membrane anchor derived from vsv-g (c) for example, or by fusion to the major capsid protein, vp39 (d). the displayed proteins are directed to the surface of the recombinant baculovirus-infected insect cells using a signal sequence derived from the major acmnpv envelope protein gp64 (gp64ss) for example. genes encoding the fusion proteins are then coupled with a strong baculoviral promoter (e.g., polh or p10) for strong expression in insect cells. the vectors can be further equipped with an expression cassette encoding a reporter or a suicide gene under a mammalian promoter (e.g., cmv or sv40), enabling transduction monitoring in mammalian cells. cell surface display can be applied in library screening for studying ligand-receptor interactions and antigen recognition. display on the viral envelope provides possibilities for transductional targeting, whereas capsid display rather facilitates studies on intracellular trafficking as well as nuclear targeting of the virus. has generally been the method of choice for altering the baculovirus tropism. boublik et al. (1995) were the first to demonstrate display of foreign proteins on the surface of baculovirus analogous to the established prokaryotic phage display systems. glutathione-s-transferase was used to construct several fusion variants with the gp64 gene. in addition, the hiv-1 major surface glycoprotein, gp120, was successfully incorporated to the amino terminus of gp64, illustrating functional ligand-binding activity when displayed on the viral surface (boublik et al., 1995) . this report was followed by a study where another hiv-1derived protein, the ectodomain of gp41, was coupled with both the native and truncated (membrane anchor) forms of gp64 (grabherr et al., 1997) . to diversify the growing collection of heterologous gp64 fusion constructs, mottershead et al. (1997) published a novel report where the green fluorescent protein (gfp) of aequorea victoria or rubella virus spike proteins e1 and e2 were displayed for the first time on the surface of an enveloped virus as n-terminal fusions to gp64. together, these initial reports illustrated that gp64 is a suitable fusion partner for functional display of foreign polypeptides on the viral surface, providing growing possibilities for viral vector targeting. next, the gp64 display strategy was expanded to include presentation of targeting moieties, that is, murine and human single chain antibody fragments (scfv) for the hapten 2-phenyloxazolone and carcinoembryonic antigen (cea), respectively, and synthetic igg-binding z/zzdomains of protein a (mottershead et al., 2000) . the viruses exhibited strong binding ability to the corresponding antigens and intact antibodies in vitro, demonstrating that the characteristics of the displayed polypeptides were preserved when presented on the viral surface. to achieve targeted baculovirus transduction, the viruses displaying zz-domains or scfv specific for the cea were further modified to include a dual expression cassette containing gfp and enhanced green fluorescent protein (egfp) under the transcriptional regulation of the polyhedrin and cmv promoters, respectively, allowing monitoring of transgene expression in both insect and mammalian cells (ojala et al., 2001) . despite improved binding, enhanced transgene expression was not observed. a baculovirus displaying an integrin-specific motif, rkk, as a part of two different loops of gfp fused with the gp64 was shown to bind a peptide representing the receptor-binding site of an 2 integrin, the 2i domain, by elisa. again, this interaction was not strong enough to overcome binding of wild-type gp64 to unknown cellular receptor(s) on the surface of 2 integrin-expressing chinese hamster ovary (cho) cells or to improve virus uptake (riikonen et al., 2005) . thus, althou gh en hanced vira l binding to desire d targets has been achiev ed, this interac tion has gene rally not led to impr oved in ternalization and gene transd uction of the vectors . in a stud y, however, th e avidin -biotin techn ology was used for baculov irus targetin g, result ing in both en hanced and targ eted transd uction ( raty et al. , 2004 ) . due to its posit ive charg e at physiolo gical ph, avid in itsel f was demonstrated to enha nce vira l transd uction of rat malignan t glioma cells (bt4c) and rab bit aortic smoo th muscle (raasm c) cells by 5-and 26-fol d, respectivel y. moreo ver, chimer ic avidin-g p64 enable d viral targeting and efficie nt gene transf er to biotiny lated cells, possibly providi ng a versati le tool for gene delivery. the display of v 3integrin -specific rgd motifs, deriv ed from the c-term inus of cox sackievirus a9 or huma n parech ovirus 1 vp prot ein, on the vir al surf ace, result ed in bo th impr oved bi nding and, thus, enhan ced transdu ction of human lung carcino ma cells expres sing v 3-integ rins (erns t et al. , 2006; matilain en et al. , 2006 ) . althou gh foreign prot ein sequence s have mainly been displa yed on the viral surf ace after fusio n to gp64, heterol ogous viral glyco prot eins are capable of serving in the same context. as a model syste m, a truncat ed form of the vesicul ar stomatitis virus glyco protein (v sv-g) was disc overed to enh ance display and en abled scattered distribution of the egfp fusio n prot eins on the vir al en velope ( chapp le and jones, 2002 ), wher eas gp 64 fusions normally accumul ate on ly at the pole of the vir ion. to apply this fusio n str ategy to bacu lovirus targeting , the vsv-g transm embrane anch or, comp rising 29 amino acids of th e cytoplas mic domain, the 20-amino acid membr ane spannin g region in additi on to the 21-amino acid truncat ed ectodoma in, was fuse d with the igg-bin ding zz domain s of prote in a ( ojala et al. , 2004 ) and displa yed on the surf ace of bac ulovirus vectors . the zz-displa ying viruses show ed improv ed binding to igg and, in princi ple, th ese vectors could be targeted to any desir ed cell type when a suitable igg antibody is available, eliminating the need of preparing distinct vectors for each application. improved transduction was not observed, however, when gfp was used as a reporter. to gain cancer cellselective tropism of baculovirus, the lyp-1 , f3 , and cgkrk (hoffman et al., 2003) tumorhoming peptides were displayed on the surface of baculovirus by fusion to the membr ane -anchor ing signal of vsv-g . to increase the specificity, the vsv-g fusion strategy was further modified by excluding the 21-amino acid vsv-g ectodomain, known to mediate nonspecific binding and transduction of the baculovirus vectors (kaikkonen et al., 2005; ojala et al., 2004) . these vectors exhibited significantly improved binding and transgene delivery to both human breast carcinoma and hepatocarcinoma cells, highlighting the potential of targeted baculovirus vectors in cancer gene therapy. in addition to vsv-g, a class i transmembrane protein, the membranespanning region of a class ii membrane protein, neuraminidase a of influenza virus, is capable of serving as an n-terminal anchor domain for efficient display of egfp on the viral surface (borg et al., 2004) , providing an alternative display strategy to gp64 or vsv-g membrane anchors. analogous to surface display, a novel baculovirus capsid display method has been developed (kukkonen et al., 2003; oker-blom et al., 2003) . this technique is based on the display of foreign proteins or peptides on the surface of the viral capsid as amino or carboxy terminal fusions to the major acmnpv capsid protein vp39 (thiem and miller, 1989a,b) . in the first capsid display report, vp39 was demonstrated to be compatible for incorporation of a foreign protein molecule, egfp, in large quantities. egfp was successfully fused either to the n-or c-terminus of vp39 without compromising the viral titer or functionality (kukkonen et al., 2003) . in addition, it was proposed that the block in transduction of mammalian cells by baculovirus lies in the cytoplasmic trafficking or nuclear import instead of viral escape from the endosomes, as had previously been suggested. thus, this new tool provides possibilities for specific intracellular and nuclear targeting of the viral capsids, and facilitates baculovirus entry and nuclear import studies in both insect and mammalian cells. it is now evident that heterologous targeting moieties with specific and high avidities can be functionally displayed in large quantities on both the baculovirus envelope and capsid. although targeting appears to be an attractive concept to enhance baculoviral transduction of specific cell types, its applicability in human disease could be partly limited by the fact that mammalian host cell nonpermissiveness cannot always be reversed simply by making baculoviral binding and entry possible. therefore, tissue targeting and nuclear localization signals could be displayed in different combinations on the viral envelope and nucleocapsid, respectively, enabling both cellular and nuclear targeting. in addition to appropriate targeting molecules, introduction of tissue-specific promoters and complement resistance (huser et al., 2001) into the baculovirus vectors could enable targeting of this insect virus to desired cells and tissues in vivo and therefore provide potential applications in gene therapy. pseudotyping, that is, phenotypic mixing, is a process in which the natural envelope glycoproteins of the virus are modified, replaced, or expressed with surface (glyco)proteins from a donor virus. in this way, the host range of virus vectors can be expanded or altered. if successful, such particles possess the tropism of the virus from which the protein was derived. the vesicular stomatitis virus (vsv) g-protein (vsv-g) is among the first and still most widely used glycoprotein for pseudotyping viral vectors due to the very extensive tropism and stability of the resulting pseudotypes. generation of vsv-g pseudotypes from a number of viruses has been described earlier. both native and modified vsv-g have been extensively used for pseudotyping retroviruses (croyle et al., 2004; emi et al., 1991; guibinga et al., 2004; schnitzer et al., 1977) , adenoviruses (yun et al., 2003) , and herpesviruses (anderson et al., 2000; tang et al., 2001) , for example. in the case of vsv-g-pseudotyped viral vectors where tissue targeting through ligand incorporation into vsv-g has been endeavored, several factors including the lack of three dimensional crystal structure of the glycoprotein, has rendered the tropism modification challenging. regardless, permissive epitope/ligand insertion sites have been identified within native vsv-g that allow modification of the protein without compromising folding or oligomerization (guibinga et al., 2004) . in addition, the use of different recombinant vsv vectors for vaccine production has been broadly studied (mckenna et al., 2003; schlehuber and rose, 2004) and different truncated forms of vsv-g have served as partners for constructing chimeric fusion proteins to facilitate the study of the biological properties of viral or cellular membrane glycoproteins (basu et al., 2004; buonocore et al., 2002; lagging et al., 1998; schnell et al., 1996) . while the vsv-g-pseudotyped vectors are valuable for many diverse studies and even for some preliminary clinical applications, their promiscuous susceptibility for target cells and tissues may contribute to toxicity and serious adverse effects through transduction of nontarget cells (burns et al., 1993; naldini, 1999; vandendriessche et al., 2002) . pseudotyped baculovirus vectors engineered to date represent viral particles bearing heterologous glycoproteins on their envelope, similar to other virus vectors, mainly the vsv-g, expressed either alone or with the endogenous baculovirus surface glycoprotein, gp64 (barsoum et al., 1997; facciabene et al., 2004; kitagawa et al., 2005; mangor et al., 2001; park et al., 2001; pieroni et al., 2001; tani et al., 2001 tani et al., , 2003 . the primary objective of these studies was to engineer vectors possessing a wider tropism and improved transduction capacity of the target cells as compared to wild-type baculovirus. secondarily, the vsv-g is expected to provide protection for baculovirus vectors against complement inactivation in potential in vivo gene therapy applications as has previously been demonstrated for vsv-g-pseudotyped retroviral vectors (ory et al., 1996) . barsoum et al. (1997) demonstrated that acmnpv can be pseudotyped with an envelope glycoprotein derived from another virus. the gene encoding vsv-g was placed under the transcriptional control of the polyhedrin promoter, providing abundant expression in infected insect cells and subsequent incorporation into budded virions, which exhibited atypical oval-shaped morphology and occasionally tail-like structures. however, no further studies have been published where the effect of vsv-g on the morphology of the budded form of acmnpv has been described. these pseudotyped viruses improved transduction of hepg2 cells tenfold and also augmented transgene delivery to certain established as well as primary cell lines that are weakly or not susceptible to transduction by wild-type baculovirus, thus broadening the tropism. in addition, it was speculated that the vsv-g may augment the escape of the virus from intracellular vesicles via its membrane fusion activity rather than improve viral binding or entry into target cells, hence escalating transport of the viral genome into the nucleus (barsoum et al., 1997) . later, vsv-g and mouse hepatitis virus s protein (mhv-s)-pseudotyped baculovirus vectors were employed as a control system in a study where cell surface components involved in baculovirus infection of insect cells and entry into mammalian cells was explored using baculovirus displaying two copies of gp64 on the viral envelope (tani et al., 2001) . it was demonstrated that the virus overexpressing gp64, in addition to its endogenous copy of gp64, can incorporate 1.5to 2-fold the normal quantity of gp64 on the budded virion. these modified viruses mediated transduction resulting in 10-to 100-fold increased reporter gene expression in a variety of cell lines as compared to the virus carrying an ordinary amount of gp64. it was also proposed that cell surface phospholipids provide a docking point for gp64, hence assisting viral entrance to mammalian cells (tani et al., 2001) . park et al. (2001) combined tropism modification of baculovirus with transcriptional targeting, designed to be limited to cells of hepatic origin. accordingly, a vsv-g-pseudotyped virus, harboring an expression reporter (luciferase) gene placed under the control of a hepatocytespecific afp (-fetoprotein) promoter/enhancer, was generated. the virus was able to transduce human hepatoma cells at an efficiency of approximately fivefold greater than the control virus lacking vsv-g and transgene expression was restricted to cells of hepatic origin expressing afp, of which concentration is elevated in hepatocellular carcinomas. the vsv-g is capable of complementing the function of gp64 by restoring the ability of a gp64-null virus to assemble and produce infectious budded virions, although the kinetics of infection is somewhat delayed and viral titers reduced by 1 to 2 logs as compared to wild-type acmnpv (mangor et al., 2001) . however, these gp64-null vsv-g-pseudotyped virions were not tested for transduction of vertebrate cells, thus, whether they could enhance transduction analogous to recombinant vectors coexpressing gp64 and vsv-g remains unanswered. in addition to vsv-g, the function of a gp64-deleted acmnpv has been partially restored by inserting the recently identified f-proteins from two group ii nucleopolyhedroviruses (npvs), lymantria dispar mnpv and spodoptera exigua mnpv, into the gp64 locus, demonstrating that f-proteins derived from heterologous npvs are functional analogs of gp64 (lung et al., 2002) . parallel to the vsv-g/gp64-null virus (mangor et al., 2001) , infectious viral titers of the f-protein pseudotypes were somewhat compromised as compared to the wild-type counterpart, suggesting that the level of compatibility between the f-proteins and other acmnpv proteins may not be optimal. further, the capacity of these f-protein-pseudotyped vectors for gene transduction of mammalian cells remains to be explored. the efficiency of gene delivery in vivo has also been explored using vsv-g-pseudotyped baculovirus vectors. the modified virus enhanced transgene delivery by five-to tenfold when mouse myoblasts and myotubes were transduced in vitro (pieroni et al., 2001) . similarly, the same increase in reporter gene (-galactosidase) expression was detected in vivo after injection of the vsv-g-pseudotyped vector in the quadriceps of balb/c and c57bl/6 mice. moreover, expression of the transgene, mouse erythropoietin, was monitored to last for 35 and 178 days in the skeletal muscle of balb/c or c57bl/6, and dba/2j mice, respectively (pieroni et al., 2001) . the vsv-g-coated baculovirus also exhibited improved resistance to inactivation by human, rabbit, guinea pig, hamster, and mouse, but not rat sera (tani et al., 2003) . this modified virus could also be used for transduction of the cerebral cortex and testis of mice by direct inoculation in vivo. no comparisons were conducted, however, with the unmodified virus to evaluate putative enhancement in transduction efficiency. a truncated form of vsv-g (vsv-ged), composed of the cytoplasmic and membrane-spanning domains in addition to the 21-amino acid ectodomain, was shown to enhance transduction by the vsv-ged-pseudotyped baculovirus both in vitro and in vivo (kaikkonen et al., 2005) . thus, the enhancement of virus transduction, which is characteristic of full-length vsv-g, was retained by the truncated form. it was speculated that the improved gene delivery was due to possible augmentation of gp64-mediated release from endosomes during viral entry into the target cells. moreover, induction of humoral and cell-mediated immune response has been studied with a recombinant baculovirus vector displaying vsv-g on the viral surface and expressing hepatitis c virus glycoprotein, e2, under the cmv promoter. the results demonstrated that cell-mediated immunity to the e2 antigen can be elicited in mice by injecting recombinant baculovirus vectors expressing the target antigen and that the display of vsv-g on the viral surface increases the immunogenic efficiency tenfold leading to greater induction of e2 antigen-specific cd8 þ t cells (facciabene et al., 2004) . ligand-directed gene delivery was achieved by pseudotyped gp64-deleted baculovirus vectors carrying measles virus receptors, cd46 and slam, on their surface (kitagawa et al., 2005) . the viruses were able to replicate and spread infection in gp64-complementing sf9 cells, whereas virus propagation was strongly reduced in cells not expressing gp64. however, after three rounds of passage of the pseudotyped viruses, the gp64-coding gene was integrated into the baculovirus genome probably through nonhomologous recombination. the corresponding viruses were able to target gene delivery to bhk cells expressing the measles virus h and f envelope glycoproteins and the transduction could be inhibited by pretreatment with specific monoclonal antibodies for the displayed ligands. a short hairpin rna (shrna) delivery system mediated by a vsv-g-displaying baculovirus vector was generated, resulting in knock down of an endogenous reporter gene, egfp, and suppression of porcine reproductive and respiratory syndrome virus replication in tissue culture (lu et al., 2006) , highlighting the potential of recombinant baculovirus as an alternative vehicle for antivirus shrna delivery. overall, the acmnpv-pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of heterologous viral or cellular membrane proteins as well as enabling diversification or constraint of the viral tropism. the selection of cell surface components during virus assembly in infected insect cells is flexible enough to allow incorporation of unrelated membrane proteins into baculovirus particles, yet specific enough to exclude the bulk of host proteins. the first proofs of the principle were the vsv-gpseudotyped (gp64-null) baculovirus vectors, which retained their ability to replicate in insect cells and transduce a large collection of mammalian cells. vsv-g may use common cell surface determinants as putative receptor molecules, rendering the vsv-g-pseudotyped baculovirus vectors inappropriate for cell-specific gene delivery, but ideal in applications where a limited tropism is not required. thus, such pseudotyped vectors would be particularly suitable for ex vivo gene therapeutic applications where there is no risk of transducing nontarget cell populations. the introduction of ligands with high and specific avidity into the viral envelope, as demonstrated by kitagawa et al. (2005) , could also enable baculovirus targeting in vivo. for generation of antibodies by traditional procedures, the protein or peptide is produced in a system of choice and subsequently purified before immunization. this is often cumbersome, and more importantly, the final product may not be correctly folded-an essential requirement for an adequate immune response in the host, and thereby, for generation of functional antibodies. in addition to recombinant proteins, several other systems including phage display, dna-based immunization, as well as recombinant viral infections and/or fusions to viral proteins are available for generation of antibodies. here, examples from the literature are presented where baculovirus surface display has been employed for generation of functional monoclonal antibodies against proteins of different origin. several reports also show clear evidence that display of the immunogen on the viral surface can elicit protective immune responses against viral or parasite infections by using animal models. to produce monoclonal antibodies against the human nuclear receptors lxr and fxr, the n-terminal domains of these antigens were displayed on the baculoviral surface by inserting the corresponding coding sequences between the signal sequence and the mature domain of gp64 of acmnpv (lindley et al., 2000) . this study illustrated that baculovirus display is a versatile tool applicable for antigen presentation and for rapid production of functional monoclonal antibodies once the antigen-coding sequence is available (lindley et al., 2000) . similarly, monoclonal antibodies against human peroxisome proliferatoractivated receptors (ppars) using baculovirus display have been generated. the amino terminal sequences of human ppard and pparg2 were placed at the n-terminus of gp64 and antibodies were raised by immunization with whole virus without prior purification of the immunogens (tanaka et al., 2002) . the antibodies generated by this method were functional in a variety of techniques such as immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays. antigenic epitopes of theileria parva, an intracellular protozoan parasite that causes east coast fever, a severe lymphoproliferative disease in cattle, were also successfully presented on the surface of acmnpv by adopting the gp64 n-terminal fusion strategy (kaba et al., 2003) . this approach was applied because previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes had not resulted in correctly folded protein molecules. further, a small, immunodominant antigenic site (site a) and the large polyprotein (p1) coding for the four structural proteins of foot-and-mouth disease virus (fmdv) have been displayed on the membrane of infected insect cells and consequently on the baculoviral surface by fusion to the n-terminus of gp64 (tami et al., 2000) . later, the investigators have shown that these fmvd antigens were able to elicit a specific immune response against fmvd in mice (tami et al., 2004) . similarly, yoshida et al. (2003) have shown that the rodent malaria plasmodium berghei circumsporozoite protein (pbcsp) displayed on the surface of baculovirus as a fusion to gp64 protects mice against a malaria sporozoite infection. urano et al. (2003) used an alternative approach of exploiting the baculovirus for monoclonal antibody production by displaying an integral er membrane protein scap on the extracellular, budded form of the virus. thus, scap was not displayed as a fusion to baculovirus specific proteins. scap is known to be involved in cleavage of sterol element-binding protein-2, hence its function is tightly coupled to cholesterol regulation (urano et al., 2003) . other membrane receptors, such as the -adrenergic receptor (loisel et al., 1997) and the leukotriene b4 receptor (blt1) residing on the plasma membrane have also been functionally displayed in the same context. in addition to acmnpv, bombyx mori npv (bmnpv) has been modified to display immunogens with similar aims as described earlier. rahman et al. (2003) displayed the immunodominant ectodomains of the fusion glycoprotein (f) of peste-des-petitis-ruminants virus (pprv) and the hemagglutinin protein (h) of rinderpest virus (rpv), on budded virus particles. the strategy was identical, in that the antigens were fused to gp64 of bmnpv and expressed under transcriptional regulation of the polyhedrin promoter. the investigators showed that the antigenic epitopes were properly displayed and that the recombinant virions were able to induce an immune response in mice against both pprv and rpv. finally, chang et al. (2004) aimed to produce a recombinant baculovirus that mimics severe acute respiratory syndrome corona virus (sars-cov) in its host range and infection mechanism. a baculovirus displaying a 688-amino acid fragment of sars-cov s glycoprotein as a gp64 fusion was generated and then used to examine the effect on the il-8 release in a549, nci-h520, hfl-1, and mrc-5 cells (chang et al., 2004) . together, these reports provide convincing evidence that baculovirus can be used for the functional display of heterologous proteins on its surface through budding from the infected insect cell. consequently, the baculovirus-displayed immunogens have been used to elicit immune responses needed for production of monoclonal antibodies and/ or to protect the animal host against a viral or parasite infection. display on the surface of bacteriophage is currently the most widespread method for display and selection of large collections of antibodies. this approach is robust, simple to use and, in addition, highly versatile. the selection procedures can be adapted to many specific conditions including selections on whole cells, tissues, and even animals. originally, generation of eukaryotic cdna libraries was based on plasmid vectors capable of replicating in particular eukaryotic cell types. during the last decade, however, a variety of display methods and other library-screening techniques have been under study for isolating monoclonal antibodies from collections of recombinant antibody fragments. the development of virus-based cdna expression libraries has offered several advantages over nonviral vectors regarding host cell tropism, transduction efficiency, stability of transgene expression, and production of the vector in high quantities. in 1997, granziero et al. (1997) aimed to develop a rapid method for generating baculovirus-based cdna expression libraries for screening cell surface molecules, for which antibodies are available beforehand and whose expression pattern is restricted to particular cell types. the first proof of principle was gained by cloning a cdna pool, reverse transcribed from human placenta, into the baculovirus genome and sorting the virus-infected insect cells by flow cytometry using monoclonal antibodies of an unknown specificity as probes. by this method, single positive cells could be sorted and viruses carrying the cdnas encoding the cell surface epitop es isolat ed. the first demonstr ation of using bac ulovirus disp lay for generation and screenin g of expres sion was described by ernst et al. (19 98 ) . an hiv-1 gp4 1 epitop e (e ldkwa), specifi c for th e ne utralizing hum an mab 2f5, was in serted into th e antig enic site b of infl uenza virus a hemag glutinin , and expres sed on the surface of baculov irus-infec ted insect cells . the epitop e was displayed in a library form , such that ea ch clone con tained different amino acids ad jacent to th e epitop e. thus , the purpo se of the experiment was to al ter the str uctural en vironment so th at the corre sponding epitop e would be presented in the most acces sible way, lea ding to an increas ed binding capacit y of the mab. the library consis ted of 8000 varia nts out of whic h one clone showed an increas ed spec ific binding capacit y when screened by fluo rescen ce activated cell sort ing (erns t et al. , 1998 ) . lat er, the group also described a syste m wher e the same epitop e as well as the biotin mimic str eptag ii were inserted at position 278 o f gp6 4 (erns t et al. , 2000 ) . the fact that the insertion s in to th e coding sequence of the maj or en velope protein of the vir us did not alter virus prop agat ion may be of value in furthe r developm ent of disp lay libraries . crawf ord et al. (2004) have desc ribed the use of baculov irus-infec ted insect cells as a display platf orm for class ii major hi stocomp atibility comp lex (mh cii) molec ules cov alently bound to a li brary of potent ia l pept ide mimo topes. the sequ ence encoding the peptide was embedd ed with in the genes for the mhc molecu le in the viral geno me. ther eby, each in sect cell in fected wit h a vir us particle from a library coding for different peptides , disp layed a uni que pept ide-mhc comp lex on its cellular membra ne. crawf ord et al. (2004 ) were able to identif y such pept ide mimo tope-m hc comp lexes that bound to the soluble recep tors and stimulating t cells bearing the same receptors by "fishing" with fluorescent, soluble t cell receptors. these findings should, therefore, have implications for the relative importance of peptide and mhc in t cell receptor-ligand recognition. later, the same group used this baculovirus-based display system for identification of antigen mimotopes for mhc class i-specific t cells (wang et al., 2005) . here, a mouse mhc class i molecule was displayed on the surface of baculovirusinfected insect cells with a 9-to 10-mer peptide library tethered to the n-terminus of beta2 microglobulin via a flexible linker. although there are relatively few studies on libraries generated by using baculovirus/ insect cell technology, the present examples clearly show that this technology has potential and interest in further development and utilization of this technology will likely increase. in this chapter, we have given a "state of the art" overview of strategies and technologies developed for display of foreign peptides and/or proteins on the surface of baculovirus-infected insect cells and budded baculovirus particles. data on virus targeting and transgene expression in mammalian cells, and on the generation of libraries for studying molecular recognition and protein-protein interactions using these techniques were summarized. production of monoclonal antibodies by utilization of these techniques and the benefits of using baculovirus display to elicit protective immune responses in animal models were reviewed. together, these studies show the potential for baculovirus within these areas of research and illustrate that further development and broadening of the interdisciplinary applications of this versatile and unique insect virus are justified. pseudotyping of glycoprotein d-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein g enables mutant virus attachment and entry efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus g glycoprotein the hypervariable region 1 of the e2 glycoprotein of hepatitis c virus binds to glycosaminoglycans, but this binding does not lead to infection in a pseudotype system baculovirus gp64 envelope glycoprotein is sufficient to mediate ph-dependent membrane fusion amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase eukaryotic virus display: engineering the major surface glycoprotein of the autographa californica nuclear polyhedrosis virus (acnpv) for the presentation of foreign proteins on the virus surface baculovirus-mediated gene transfer into mammalian cells characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins vesicular stomatitis virus g glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells induction of il-8 release in lung cells via activator protein-1 by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions non-polar distribution of green fluorescent protein on the surface of autographa californica nucleopolyhedrovirus using a heterologous membrane anchor mimotopes for alloreactive and conventionaltcells ina peptide-mhc display library pegylation of a vesicular stomatitis virus g pseudotyped lentivirus vector prevents inactivation in serum optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells pseudotype formation of murine leukemia virus with the g protein of vesicular stomatitis virus baculovirus surface display: construction and screening of a eukaryotic epitope library improving baculovirus transduction of mammalian cells by surface display of a rgd-motif expanding baculovirus surface display. modification of the native coat protein gp64 of autographa californica npv baculovirus vectors elicit antigen-specific immune responses in mice expression of foreign proteins on the surface of autographa californica nuclear polyhedrosis virus baculovirus cdna libraries for expression cloning of genes encoding cell-surface antigens ligand-modified vesicular stomatitis virus glycoprotein displays a temperaturesensitive intracellular trafficking and virus assembly phenotype highly efficient baculovirusmediated gene transfer into rat chondrocytes efficient gene transfer into human hepatocytes by baculovirus vectors progressive vascular changes in a transgenic mouse model of squamous cell carcinoma monoclonal antibodies to baculovirus structural proteins: determination of specificities by western blot analysis incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors biosynthesis and processing of the autographa californica nuclear polyhedrosis virus gp64 protein baculovirus surface display of theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes truncated vesicular stomatitis virus g protein improves baculovirus transduction efficiency in vitro and in vivo ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses recombinant baculoviruses as mammalian cell gene-delivery vectors baculovirus as versatile vectors for protein expression in insect and mammalian cells baculovirus capsid display: a novel tool for transduction imaging a tumor-homing peptide with a targeting specificity related to lymphatic vessels functional role of hepatitis c virus chimeric glycoproteins in the infectivity of pseudotyped virus production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles suppression of porcine arterivirus replication by baculovirus-delivered shrna targeting nucleoprotein trends in the development of baculovirus expression vectors pseudotyping autographa californica multicapsid nucleopolyhedrovirus (acmnpv): f proteins from group ii npvs are functionally analogous to acmnpv gp64 enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides a gp64-null baculovirus pseudotyped with vesicular stomatitis virus g protein membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates a combinatorial g protein-coupled receptor reconstitution system on budded baculovirus. evidence for galpha and galphao coupling to a human leukotriene b4 receptor baculovirus entry into human hepatoma cells rgd motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells recombinant rhabdoviruses as potential vaccines for hiv-1 and other diseases baculoviruses as gene expression vectors baculoviruses for foreign gene expression in insect cells insect baculoviruses: powerful gene expression vectors the gp64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection baculoviral display of the green fluorescent protein and rubella virus envelope proteins baculoviral display of functional scfv and synthetic igg-binding domains in vivo gene delivery by lentiviral vectors specific binding of baculoviruses displaying gp64 fusion proteins to mammalian cells improved display of synthetic igg-binding domains on the baculovirus surface baculovirus display strategies: emerging tools for eukaryotic libraries and gene delivery requirement for gp64 to drive efficient budding of autographa californica multicapsid nucleopolyhedrovirus the baculovirus gp64 envelope fusion protein: synthesis, oligomerization, and processing a stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus g pseudotypes hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein in vivo gene transfer in mouse skeletal muscle mediated by baculovirus vectors an analysis of the role of the target membrane on the gp64-induced fusion pore a fragment of the hmgn2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo baculovirus display of fusion protein of peste des petits ruminants virus and hemagglutination protein of rinderpest virus and immunogenicity of the displayed proteins in mouse model enhanced gene delivery by avidin-displaying baculovirus functional display of an alpha2 integrin-specific motif (rkk) on the surface of baculovirus particles prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses baculovirus replication in a mosquito (dipteran) cell line production of human beta interferon in insect cells infected with a baculovirus expression vector altering the surface properties of baculovirus autographa californica npv by insertional mutagenesis of the envelope protein gp64 presentation of antigenic sites from foot-and-mouth disease virus on the surface of baculovirus and in the membrane of infected cells immunological properties of fmdv-gp64 fusion proteins expressed on sf9 cell and baculovirus surfaces the generation of monoclonal antibodies against human peroxisome proliferatoractivated receptors (ppars) helper virus-free hsv-1 vectors packaged both in the presence of vsv g glycoprotein b support gene transfer into neurons in the rat striatum characterization of cell-surface determinants important for baculovirus infection in vitro and in vivo gene delivery by recombinant baculoviruses a baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus autographa californica nuclear polyhedrosis virus a novel method for viral display of er membrane proteins on budded baculovirus baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids oncoretroviral and lentiviral vector-mediated gene therapy using a baculovirus display library to identify mhc class i mimotopes identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus autographa californica nuclear polyhedrosis virus mechanism of neutralization of budded autographa californica nuclear polyhedrosis virus by a monoclonal antibody: inhibition of entry by adsorptive endocytosis baculovirus virions displaying plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection dl-vsvg-lacz, a vesicular stomatitis virus glycoprotein epitope-incorporated adenovirus, exhibits marked enhancement in gene transduction efficiency key: cord-346554-a98pjtxs authors: uddin, md bashir; lee, byeong-hoon; nikapitiya, chamilani; kim, jae-hoon; kim, tae-hwan; lee, hyun-cheol; kim, choul goo; lee, jong-soo; kim, chul-joong title: inhibitory effects of bee venom and its components against viruses in vitro and in vivo date: 2016-11-26 journal: j microbiol doi: 10.1007/s12275-016-6376-1 sha: doc_id: 346554 cord_uid: a98pjtxs bee venom (bv) from honey bee (apis melifera l.) contains at least 18 pharmacologically active components including melittin (mlt), phospholipase a(2) (pla(2)), and apamin etc. bv is safe for human treatments dose dependently and proven to possess different healing properties including antibacterial and antiparasitidal properties. nevertheless, antiviral properties of bv have not well investigated. hence, we identified the potential antiviral properties of bv and its component against a broad panel of viruses. co-incubation of non-cytotoxic amounts of bv and mlt, the main component of bv, significantly inhibited the replication of enveloped viruses such as influenza a virus (pr8), vesicular stomatitis virus (vsv), respiratory syncytial virus (rsv), and herpes simplex virus (hsv). additionally, bv and mlt also inhibited the replication of non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). such antiviral properties were mainly explained by virucidal mechanism. moreover, mlt protected mice which were challenged with lethal doses of pathogenic influenza a h1n1 viruses. therefore, these results provides the evidence that bv and mlt could be a potential source as a promising antiviral agent, especially to develop as a broad spectrum antiviral agent. massive growth in human population, immense rise in urbanization, drastic changes in global environment, and improved connectivity worldwide in terms of better transportation facilities have led to the emergence and re-emergence of viral diseases in human population (li et al., 2011) . outbreaks of many rna viruses are still the leading cause of morbidity and mortality in worldwide population. many antiviral agents against diverse viruses have been reported. however, effective antiviral agents which specifically target to some rna viruses such as sars-cov, influenza a (h5n1, h1n1) and measles are in shortage due to genetic variations of the virus and the lack of approved or universally recommended therapies (wen et al., 2003; lorin et al., 2005; triggiani et al., 2006) . hence, there is a global requirement for continued development of new antiviral agents, especially from natural sources to provide several alternatives for the control, prevention, and management of the spread of diseases caused by rna and dna viruses. number of compounds derived from natural sources are reported to have numerous biological activities, thus considered potential sources of various modern pharmaceuticals including antiviral agents (jacobs and coyne, 2013; kim et al., 2016; weeratunga et al., 2016) . bee venom (bv) of the honey bee apis melifera has been utilize as a traditional medicine for the treatment of rheumatism, arthritis, skin diseases, cancerous tumors, and back pains (billingham et al., 1973; son et al., 2007) due to its antibacterial, antiviral and anti-inflammatory effects (habermann, 1972; son et al., 2007; hwang et al., 2015) . such bio activities are explained by the variety of components contain in the bv such as peptides melittin (mlt), adolapin, apamin, and mast cell degranulating peptide; several enzymes including phospholipase a2 (pla2); biologically active amines (histamine and epinephrine); and non-peptide components (carbohydrates, lipids, and free amino acids) (lariviere and melzack, 1996; park et al., 2004; son et al., 2007) . however, antiviral properties of bv have not well investigated and also its mechanisms are not fully understood. in the present study, we investigated the antiviral activity of bv and its components against enveloped and non-enveloped viruses including rna and dna viruses in vitro and attempted to understand the mechanism of this property is mainly owing to the virucidal effect of both bv and mlt. additionally, we evaluated the antiviral activity of mlt against lethal doses of pathogenic influenza a h1n1 viruses in mice in vivo. bv and mlt were supplied by chung-jin biotech co. ltd, korea and compounds were dissolved in phosphate buffered saline (pbs). stock solutions were aliquoted and stored at -20°c. human embryonic kidney; hek293t (atcc ® crl-11268 tm ), human epithelial cervix adenocarcinoma; hela (atcc ® ccl-2tm), ceropithecus aethiops epithelial kidney; vero (atcc ® ccl-81), human epithelial hela contaminant carcinoma; hep-2 (atcc ® ccl-23 tm ) and canis familiaris kidney epithelial; mdck (atcc ® ccl-34 tm , nbl-2) cells were grown and maintained in dulbecco's modified eagle's medium (dmem) (invitrogen) supplemented with 10% fetal bovine serum (fbs) (gibco) and 1% antibiotic-antimycotic solution (gibco) . all cell cultures were incubated at 37°c in a humidified atmosphere supplied with 5% co2. green fluorescent protein (gfp)-fused vesicular stomatitis virus (vsv-gfp) and herpes simplex virus (hsv-gfp) were propagated on confluent vero cells, and enterovirus-71 (ev-71) and gfp-fused coxsackie virus (h3-gfp) were propagated on confluent hela cells. gfp-fused respiratory syncytial virus (rsv-gfp) was propagated on hep-2 cells. gfp-fused influenza a (a/puertorico/8/34) (h1n1) (pr8-gfp) and challenge viruses of influenza a subtype (a/pr/8/34) (h1n1) were propagated in the allantoic fluid of 10-day-old chicken embryos. all the vsv-gfp, pr8-gfp, h3-gfp, rsv-gfp, and hsv-gfp viruses were kindly gifted from dr. j. u. jung, department of molecular microbiology and immunology, university of southern california, usa. a/pr/8/34(h1n1) was provided by dr. y.k. choi, chunbuk national university, cheongju, republic of korea. the cytotoxic effect of bv or mlt in hek293t, mdck, hep-2, vero or hela cell monolayers were determined by quantifying the cell viability using trypan blue exclusion test as described previously (strober, 2001) . after cells grown in 72well plates into a 75-80% confluent monolayer, different concentrations (0.5-10 μg/ml) of bv or mlt was added to the each well. the dilution medium without the samples was used as a control. experiment was performed in triplicate, and all the plates were incubated at 37°c in a humidified 5% co2. clarified cells were then stained with 0.4% trypan blue (invitrogen) (ratio: 1:1) at 24 h post-treatment (hpt) and mounted to the hemocytometer to obtain the percentages of viable cells. concentrations of the bv or mlt was plotted against the cell viabilities for each cell type and cytotoxicity of the bv to each cell type is expressed as the 50% cytotoxic concentration (cc 50 ), the concentration which 50% of cell death occurred relative to non-treated control cells. ec50 value of bv or mlt against various enveloped and nonenveloped viruses were determined. to determine the ec 50 of bv or mlt, hek293t, hela, vero, mdck, and hep2 cells were cultured in 12-well (1 × 10 6 cells/well) plates, and incubated at 37°c in a 5% co 2 atmosphere. after 12 h, viral suspensions were co-incubated with different concentrations of bv or mlt (0.5-10 μg/ml) at 4°c for 30 min and inoculated to the each cell types; bv and mlt: rr8-gfp (moi= 2.0; mdck), rsv-gfp (moi=1.0; hep2), hsv-gfp (moi =1.0; vero), h3-gfp (moi=2.0; hela), and ev-71 (moi =1.0; hela). ec 50 of bv and mlt for the vsv-gfp (moi =0.2) was tested in hek293t and vero cells, respectively. at 2 h post-infection (hpi), the culture medium was replaced with 10% fbs dmem media, and at 24 hpi, cells or supernatants were harvested and virus titer was evaluated as described below. the ec 50 values were calculated as the bv or mlt concentration yielding 50% reduction of titer compared to the control (virus infection without bv or mlt). the experiments were performed in triplicate. to determine the possible antiviral mechanism of bv, three different treatment groups were designed for comparison of the antiviral effect of bv: (1) bv pre-treated group: hek293t cells were pre-treated with 3.0 μg/ml of bv at 37°c for 12 h and then infected with vsv-gfp (moi=0.2). similarly 1,000 units/ml recombinant human interferon-β (rhifn-β, sigma) was treated as a positive control. as a negative controls, dmem medium only, and virus infection without bv treatment were used; (2) bv co-incubation group: hek293 cells were inoculated with mixture of 2.0 μg/ml bv and vsv-gfp (moi=0.2), which was co-incubated at 4°c for 30 min; and (3) post-treated group (virus replication inhibition after entry): hek293t cells were infected with vsv-gfp first at 37°c for 4 h, and then treated with 3.0 μg/ml of bv. to compare the antiviral effects of bv to vsv-gfp infection, the levels of viral replication (which reflected by gfp expression) were photographed (under 200x magnification) at 12 or 24 hpi of vsv-gfp, and calculated the virus titer by standard plaque assay with some modifications. briefly, vero cells were seeded in 12-well (8 × 10 5 cells/well) plates. after 12 h when cells became confluent monolayer, the harvested cell supernatants containing each replicated virus were 10-fold serially diluted and 100 μl of each dilution was added to the 1% fbs containing each wells. the media in the wells were replaced with 1% agar overlay at 2 hpi and 1-2 days later, number of plaques showing gfp were counted under the microscope (200x magnification). virus titer was calculated using the number of gfp showing plaques and the dilution factor (reed and muench, 1938; coil and miller, 2004) , and expressed as log 10 plaque forming unit/ml (log 10 pfu/ml). to determine whether bv could induce type i ifn, ifn-β secretion levels were measured in hek293t cell supernatants of 3.0 μg/ml bv or rhifn-β (positive control) or dmem medium (negative control) treated samples, which was harvested at 24 hpt. the ifn-β levels were measured using commercial human ifn-β elisa kits (tfb inc.) according to manufacturer's protocol. to determine the antiviral effect of bv or mlt, co-incubation experiments were carried out against different viruses (mlt: vsv-gfp; bv and mlt: pr8-gfp, rsv-gfp, hsv-gfp) in several cell lines. vero, hela, mdck, and hep2 cells were cultured in 12-well (8 × 10 5 cells/well) plates, and incubated at 37°c for 12 h. each viral suspensions (vsv-gfp: moi=0.2; pr8-gfp: moi=2.0; rsv-gfp: moi=1.0; hsv-gfp: moi=1.0) were co-incubated with 2.0 μg/ml of bv or mlt at 4°c for 30 min. after the incubation, the mixture were inoculated to the specified cell monolayers in 12 well plates (vero: vsv-gfp, hsv-gfp; mdck: pr8-gfp; hep2: rsv-gfp) using dmem supplemented with 1% fbs. similarly 1,000 units/ml rhifn-β and viruses were co-incubated and inoculated to the cells, as a positive control for bv antiviral activity assay. dmem medium only and virus infection without any bv or mlt treatments were used as negative controls. unabsorbed viruses and residuals were aspirated out with the supernatant at 2 hpi, and dmem supplemented with 10% fbs. the residual virus infectivity in each treated samples (which reflected by gfp expression) was photographed (under 200x magnification) at 24 hpi. cell supernatants (vsv-gfp, rsv-gfp, and hsv-gfp) or cells (pr8-gfp) were collected at 24 hpi, and expressed gfp was quantified by measuring gfp absorbance using glomax detection system (promega). viral replication levels were calculated by standard plaque assay as described. this virus titer was considered as the residual infectivity (log10 pfu/ml) to evaluate the virucidal activity of the bv or mlt against different viruses tested. bv or mlt antiviral activity was tested in hela cells with h3-gfp and ev-71 viruses using standard antiviral activity assays. h3-gfp (moi=1.0) or ev-71 (moi=1.0) viral suspensions were co-incubated at 4°c for 30 min with 2.0 μg/ml of bv or mlt. after the incubation, the mixtures were inoculated to the 12 h cultured monolayers of hela cells (6well plates; originally seeded 1 × 10 6 cells/well). rest of the antiviral assay procedures were similar to the above section. additionally, bv or mlt antiviral activity for ev-71 was determined by evaluating 50% tissue culture infective dose (tcid 50 ) and analysis of mrna expression levels of the ev-71 vp1 gene. the residual infectivity was titrated for the each samples collected at 24 hpi by tcid50, (the amount of virus required to produce cytopathic effect in 50% of inoculated cells) as described previously and expressed as log 10 tcid50/ml . moreover, ev-71 vp1 gene mrna expression levels were determined at 24 hpi. total mrna from the cells, which inoculated after co-incubation of ev-71 (moi=1.0) with bv (2.0 μg/ml) or mlt (2.0 μg/ml) was extracted using rneasy mini kit (qiagen) and cdna was synthesized using reverse transcriptase (toyobo) according to manufacturer's protocol. to determine the ev-71 mrna expression levels, ev-71 vp1 gene was amplified using gene specific primers (ev-71 vp1 forward primer; vp1fw-5 -ttgacaaaaactgaggggtt-3 and the reverse primer; vp1rev-5 -ttgacaaaaactgaggggtt-3 (wen et al., 2003) . gapdh, forward primer 5 -tgacca cagtccatgccatc-3 and the reverse primer 5 -gac ggacacattgggggtag-3 were used as a house keeping gene (seal et al., 1995) . after the real-time polymerase chain reaction (rt-pcr), equal amount of amplified products were run on 1.5% ethidium bromide agarose gels and visualized using geldoc imaging system (bio-rad). the relative vp1 gene fragment band intensity (rbi) was compared with gapdh using band quantification software (bio-rad). to find out the effective time point which mlt exhibits virucidal activity, 2.0 μg/ml of mlt and vsv-gfp (moi=0.2) were co-incubated for 5, 10, 20, and 30 min at 4°c, and then mixture was inoculated to hek293t cell monolayers in 6 well plates. dmem medium only and virus infection without any mlt treatments were used as negative controls. unattached viruses and mlt were aspirated out with the supernatant at 2 hpi, and dmem supplemented with 10% fbs was added to the wells. the virucidal effect of mlt was analyzed by observing the gfp expression under 200× magnification at 24 hpi. gfp absorbance and virus titer was quantified as described in earlier sections. to determine the effect of mlt on virus infection and the possible effect on the initial stages of viral replication cycle in vitro, following treatment groups were designed: (1) virus attachment assay: co-treatment; 2.0 μg/ml of mlt and pr8-gfp (moi=2.0) were simultaneously added to the mdck cells for 1h at 4°c. residual virus or mlt were aspirated out with the supernatant at 2 hpi, and replaced with 10% fbs dmem media; (2) entry assay: post-treatment; mdck cells were infected with pr8 (moi=2.0) first, and after 1 h of adsorption, cells were washed 3 times with pbs to remove unattached viruses and treated the cells with dmem (10% fbs) containing 2.0 μg/ml of mlt. dmem medium only and virus infection without any mlt treatments were used as negative controls. effect of mlt on virus attachment and virus entry to the cells was determined by measuring the gfp expression at 24 hpi, using glomax multi-detection system (promega). virus titer was determined as described in method section and expressed as log10 pfu/ml. sedimentation velocity ultracentrifugation (gastaminza et al., 2006) was carried out to delineate the virucidal mechanism of enveloped viruses. in here, sucrose gradient separation procedure was conducted with some modifications to identify the fractions containing the infectious viral particles and to estimate their size (molecular mass) by immonoblotting. in brief, a discontinuous sucrose density gradient was prepared by layering successive decreasing sucrose density solutions (from 20-60%) upon one another in the polyallomer tubes. once the discrete layers of sucrose was visible, pr8-gfp mixed with pbs or pr8-gfp mixed with mlt, which was co-incubated at 4°c for 30 min were applied on to the top. the tubes were ultra-centrifuged in swinging bucket sw-28 rotor (beckman) at 26,000 rpm at 4°c for 4 h. after ultracentrifugation, without disturb the sucrose layers, total 30 ml of fractions (assuming that some of the fractions containing virus particles) were collected (30 × 1 ml) from all the gradients. in brief, total 6 ml from each gradient (6 × 1 ml) was collected (3 × 1 ml from upper layer and 3 × 1 ml from lower layer from each sucrose gradient). out of these fractions, 2 nd and 5 th fractions from each gradient (2 nd and 5 th fractions of 20%, 30%, 40%, 50%, and 60% denoted sequentially from 1 to 10 in the figs.) were taken for the immunoblotting using polyclonal antibody derived from h1n1 virus infected mouse serum to identify the which fractions containing the virus particles. as an immunobloting positive control (pc), native pr8-gfp which heated at 100°c for 10 min prior to loading was used. moreover, the same fractions which used for immunobloting were examined for the infectivity of the virus particles by observing and measuring the gfp expression at 24 hpi, after infection to mdck cells. all the animal studies conducted under appropriate conditions with the approval of the institutional animal care and use committee of bioleaders corporation, daejeon, south korea, protocol number: bls-abls-13-008 and all the treatment and challenge experiments were conducted in an approved bsl-2 + laboratory facility. in this study, mouse model was chosen to determine whether mlt exhibits antiviral protection in vivo. mice survival rate and the body weight were evaluated after challenge with co-incubated mixture of pbs or mlt and influenza a virus subtype h1n1 (pbs/h1n1 or mlt/h1n1). for the whole experiment, 6-week-old 20 c57bl/6 female mice (17 ± 1 g) were used and for the survival rate analysis, 12 mice were divided into 2 groups; pbs/h1n1 treated (control) (n=6) and mlt/h1n1 treated (n=6). for the lung virus titration, another 8 mice were grouped into 2 groups; pbs/h1n1 (n=4) and mlt/h1n1 treated (n=4). first, 15 μl of mlt (100 ng) was co-incubated with 15 μl of 5´ minimum lethal dose (mld 50 ) of h1n1 for 30 min at 4°c, and then 30 μl from the mixture was intranasally administered per mice (5.9 ng/g body weight). body weight and survival rate were noted up to 8 days post-infection (dpi). mice showing more than 25% body weight loss were considered as an experimental end point and were humanely killed. lung tissues were collected aseptically from euthanized mice at 5 dpi and homogenized with equal volume (1 ml/g) of pbs containing 1% antibiotic/antimycotic solution. samples were centrifuged (10,000 × g at 4°c for 10 min), supernatants were immediately 10-fold serially diluted and inoculated into cultured mdck cells grown to confluence on 96 well tissue culture plates. the lung viral titer was calculated by the reed and muench method and expressed as log10 tcid 50 /lung . statistical analysis was performed using graphpad prism software version 6 for windows (graphpad software). all in vitro assays were carried out in triplicate and the data were expressed means ± standard deviations (sd). differences between groups were analyzed by analysis of variance (anova) or student's t-test. comparison of survival was done by logrank test using graphpad prism 6 version. p < 0.05 were regarded as significant. the different concentrations of bv was tested for all the cell types used in this study to determine whether the bv is cytotoxic to the cells. cc 50 of bv and mlt in vitro is presented in the table 1 . as shown, bv cc50 for the hek293t, hela and mdck cells were 8.61 ± 0.02, 8.98 ± 0.07, and 8.35 ± 0.08 μg/ml, respectively, representing 50% of the cells were viable at this concentration to the cells. lowest cc50 value of bv was obtained for the hep2 (6.25 ± 0.12 μg/ml) among tested cell lines followed by vero cells (7.13 ± 0.1 μg/ml). the cytotoxic concentration of mlt was also tested via tryphan blue staining and cc 50 of mlt for vero, mdck, hep2 and hela cells were 6.23 ± 0.02, 7.66 ± 0.94, 5.02 ± 0.17, and 4.36 ± 0.54 μg/ml, respectively. according to the cc50 values, mlt dose which showing more than 90% cell viability was selected for the experiments; 2.0 μg/ml or low mlt dose for vero, mdck, and hep2 cells, and 1.0 μg/ml or low mlt dose for hela cells. ec 50 of bv and mlt in vitro is presented in the table 1 . ec 50 value of bv against vsv-gfp, pr8-gfp, rsv-gfp, hsv-gfp, h3-gfp, and ev-71 were 0.5 ± 0.06, 1.81 ± 0.08, and 1.17 ± 0.09, 1.52 ± 0.11, and 0.5 ± 0.04, respectively and virus replication was inhibited by 50% at this concentrations. moreover, ec 50 of mlt was approximately 1.18 ± 0.09, 1.15 ± 0.09, 0.35 ± 0.08, 0.94 ± 0.07, 0.99 ± 0.09, and 0.76 ± 0.03 μg/ml for vsv-gfp, pr8-gfp, rsv-gfp, hsv-gfp, h3-gfp, and ev-71, respectively. the data demonstrated that ec50 of bv against vsv-gfp and h3-gfp were lower than the ec 50 values of mlt. moreover, bv exhibited higher selectivity index (si) than the mlt against vsv-gfp and h3-gfp (table 1) . considering effectiveness and cytotoxicity, 1.0 or 2.0 μg/ml of bv or mlt was used for the subsequent in vitro antiviral assays and utilized doses were mentioned in the method section where appropriate. from above, we could suggest that the antiviral activity of bv or mlt against the viruses examined, were not resulted from the cytotoxicity of the peptide on the tested cells. to determine the antiviral activity and to understand the possible antiviral mechanism of bv, three different treatments were conducted to vsv-gfp virus in hek293t cells as described in material and methods. bv (3.0 μg/ml) pre-treating at 37°c for 12 h before infecting the vsv-gfp to the cells (virus/peptide pre-treatment) showed significant reduction of virus replication at 24 hpi (354-fold), which the pattern was comparable to the rhifnβ pre-treating, compared to the only vsv-gfp infected cells (fig. 1a) . moreover, ifn-β levels were measured in the bv (3.0 μg/ml) treated cell supernatant of hek293t (fig. 1b) . bv significantly induced higher levels of secreted ifn-β compared to the control (medium only), which was similar to the pattern observed in rhifn-β treated hek293t cell supernatant. thus, data indicated that bv can mediate the antiviral innate immune responses in epithelial cells by triggering the expression of type i ifn, which may stimulate the antiviral state in the cells. as shown in fig. 1c , bv (2.0 μg/ml) co-incubating with vsv-gfp at 4°c for 30 min before inoculating to the cells (virus/peptide co-incubation) also showed prominent reduction in virus titer at 24 hpi (5,370-fold) compared to the only vsv-gfp infected cells. although in a low degree, hek293t cells infected with vsv-gfp and treated with 3.0 μg/ml of bv (post-treatment) at 4 hpi also showed inhibition of virus replication at 12 (16136-fold) or 24 hpi (63-fold) (fig. 1d ). both bv and rhifnβ post-treated cells showed similar virus titer, espe-cially at 12 hpi, and it was significantly lower than the only vsv-gfp infected cells. based on the three treatment modes carried out, we speculate that anti-viral activity of bv attributed to stimulating type i ifn signaling, virucidal activity, and ability for the inhibition of virus replication after virus enter to the cells. after investigation the anti-vsv efficacy of the bv, its effectiveness against other enveloped viruses were investigated. pr8-gfp, rsv-gfp, and hsv-gfp co-incubating with bv (2.0 μg/ml) at 4°c for 30 min before inoculating to the cells (virus/peptide co-incubation) showed reduction in the virus infectivity (fig. 2) . bv treated pr8-gfp ( fig. 2a) , rsv-gfp (fig. 2b) , and hsv-gfp (fig. 2c) showed distinct reduction in gfp expression (3-fold, 2-fold, and 7-fold, respectively) and virus titer (175-fold, 7-fold, and 9-fold, respectively) at 24 hpi compared to the untreated virus infected groups. this suggest that bv could disrupt the infectivity of broad spectrum of enveloped viruses, which further convincing its virucidal effect. . gfp expression levels were measured by gloma multi dectection luminometer (promega). the virus titers were determined by standard plaque assay. virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). after investigated the different antiviral effect of the bv, several compounds in bv were tested to find out which component of the bv responsible for its virucidal effect. among those, only mlt showed the similar effects as bv. for further confirmation, the virucidal effect of mlt against enveloped rna (vsv-gfp, pr8-gfp, and rsv-gfp) and dna (hsv-gfp) viruses were tested. in all the tested cells, there was a direct effect of the mlt to the virus which shows reduced virus infectivity (fig. 3) . different viral suspensions were coincubating with mlt (2.0 μg/ml) at 4°c for 30 min before inoculating to the cells showed marked reduction in gfp expression at 24 hpi, whereas the untreated virus infected groups showed significantly high levels of gfp expression for vsv-gfp (fig. 3a) , pr8-gfp (fig. 3b) , rsv-gfp (fig. 3c ), and hsv-gfp (fig. 3d) . in relation to this, mlt treated groups showed reduced viral supernatant titers for vsv-gfp (1598-fold), pr8-gfp (5-fold), rsv-gfp (82-fold), hsv-gfp (16-fold) compared to the only virus infected group. collectively, it suggests that mlt has the anti-viral activity by the virucidal effect against broad spectrum of enveloped viruses. after identifying that bv has virucidal effect on broad spectrum of enveloped viruses, its efficacies against non-enveloped rna viruses such as h3-gfp and ev-71 were also evaluated. h3-gfp and ev-71 viral suspensions were coincubated for 30 min at 4°c with 2.0 μg/ml bv or rhifn-β, and the mixture (bv/h3-gfp and rhifn-β/h3-gfp or bv/ ev-71 and rhifn-β/ev-71) was inoculated to the monoev-71/vp1 gene mrna expression was evaluated using vp1 gene specific pcr primers and all the samples normalized using gapdh. rbi (gene/ gapdh) was determined using geldoc imaging system band quantification software (c and f). virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). layers of hela cells. at 24 hpi, marked reduction of gfp expression was observed in bv/h3-gfp treated cells compared to untreated cells (fig. 4a) . in relation to this, significantly lower (977-fold) virus titer was detected in bv/h3-gfp treated cells than the only h3-gfp infected cells (fig. 4a) and was comparable to virus titers of the rhifn-β/h3-gfp treated cells. moreover, ev-71 induced cpe was markedly inhibited and the cell viability was higher in the bv/ev-71 treated cells than the only ev-71 infected group (data not shown). the only ev-71 infected cells showed significantly higher (131-fold) tcid 50 than bv/ev-71 treated cells (fig. 4b ). ev-71 vp1 mrna expression levels were decreased in bv/ev-71 treated cells compared to the only ev-71 infected cells by 2-fold (fig. 4c) , exhibiting lower rbi of the vp1 gene. interestingly, mlt co-incubated with h3-gfp also showed lower gfp expression (1.5-fold) and virus titers (5-fold) in comparison to the only h3-gfp infected cells (fig. 4d) . ev-71 exhibited significant reduction in cpe and high cell viability in the mlt/ev-71 treated cells than the only ev-71 infected group (data not shown). additionally, only ev-71 infected cells showed significantly higher (236-fold) tcid50 than mlt/ev-71 treated cells (fig. 4e) . moreover, ev-71 vp1 mrna expression levels were decreased in mlt/ev-71 treated cells compared to the only ev-71 infected cells by 4-fold (fig. 4f) , exhibiting lower rbi of the vp1 gene. collectively, data demonstrated that bv or mlt exerts its virucidal antiviral activity not only against enveloped viruses, but also to non-enveloped viruses. . gfp expression levels were measured by gloma multi dectection luminometer (promega). the virus titers were determined by standard plaque assay. virus titers were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). to determine the effective time needed for mlt to act on virus, 2.0 μg/ml mlt was incubated with 0.2 moi vsv-gfp at 4°c at different times. as shown in fig. 5a , infectivity of vsv-gfp was dramatically suppressed with the increasing time (5-30 min) of incubation. virus infectivity initiated to reduce from 5 min incubation and during 30 min incubation, it showed high virucidal effect similar to our previous data. this suggests that mlt can inactivate or inhibit virus replication even it was contact with the virus for a short period of time and inactivate the infectivity. to further understand the effect of mlt on viral replication cycle, virus-cell attachment and virus-entry assay were performed. as shown in fig. 5b , gfp absorbance and virus titers of mlt treated groups were not significantly different compared to the control (only virus infected) in both attachment and entry assays. these results indicate that mlt could not interfere the initial steps of the virus life cycle and unable to inhibit virus infection and replication, once the virus is infected to the cells. it evident from the data that mlt involves in the steps occurring prior to virus-cell attachment, mainly may direct interaction of the virus and mlt, and in turn inhibiting the virus infectivity. to clarify the virucidal activity of the mlt against enveloped viruses, sedimentation velocity ultracentrifugation was conducted using continuous sucrose gradient for pr8-gfp viruses co-incubated with pbs or mlt and subjected to immunoblotting as described in materials and methods. as shown in the fig. 6a , surface viral proteins such as hemagglutinnin (ha), nucleoprotein (np), and neuraminidase (na) of pr8-gfp co-incubated with pbs were detected in the 40% sucrose . 6 . virucidal mechanism study of mlt by sedimentation velocity ultracentrifugation. pr8-gfp virus was co-incubated with the pbs or mlt for 30 min at 4°c. samples were loaded on the top of the 20-60% continuous sucrose density gradient and placed into ultracentrifugation with sw-28 rotor at 26,000 rpm for 4 h in 4°c. fractions were collected and 2 nd and 5 th fractions out of six layers from each sucrose gradient were subjected to immunoblotting. 2 nd and 5 th fractions of 20%, 30%, 40%, 50%, and 60% numbered sequentially from 1 to 10. (a) pr8-gfp viral proteins (ha, na, and np) containing in each fraction was detected using polyclonal antibody derived from h1n1 virus infected mouse serum. native pr8-gfp was loaded as an immunobloting positive control (pc). (b and c) the same fractions which used for immunobloting were examined for the infectivity of the virus particles by observing and measuring the gfp expression at 24 hpi, after infection to mdck cells. gfp images were obtained under fluorescence microscopy (200x magnification). gfp expression levels were measured by gloma multi dectection luminometer (promega). gfp expression levels were expressed as mean ± sd. scale bars, 100 μm. error bars indicate the range of values obtained from triplicate in three independent experiments (*p < 0.05 and **p < 0.01 indicates a significant difference between groups compared by student's t-test). cushion (center fractions number 5 and 6), corresponding to the protein band sizes of the pr8-gfp positive control (pc). in contrast, those proteins of the pr8-gfp co-incubated with mlt were observed mainly at the upper sucrose gradient fractions (20% sucrose cushion) (number 1 and 2) followed by slightly in the 30% (fraction number 3 and 4), suggesting that some structural destabilization has occurred due to the mlt, however, no effect to the virus structural proteins. moreover, pr8-gfp viruses treated with pbs fractions (40% sucrose cushion) showed high virus infectivity in vitro, however, viruses co-incubated with mlt did not show the infectivity at over 40% sucrose cushions ( fig. 6b and c) . cells that were treated with the fractions from 20 to 30% sucrose cushion were very weakly infected, probably due to the remained intact virus particles that has not been interacted with the mlt. from the mass differences between native pr8-gfp and the mlt co-incubated with pr8-gfp fractions, it demonstrates that mlt has direct effect on structure destabilization of the virus particle, thus interrupts the virus infectivity. to evaluate the efficacy of mlt in vivo, mlt (100ng) were incubated with 5mld50 of h1n1, for 30 min at 4°c and the mixture (pbs/h1n1 or mlt/h1n1) was inoculated intranasally and monitored daily for morbidity. no toxicity was observed in mlt/h1n1 treated mice, as indicated by weight loss (fig. 7a ) and the survivability (fig. 7b) . mlt/h1n1 treated mice showed slightly decreased body weight until 2 dpi and regained its weight until 8 dpi, whereas, pbs/h1n1 treated mice exhibited severe weight loss and most of all (5mld50/mouse) mixture co-incubating for 30 min at 4°c were intranasally administrated to six-week-old c57bl/6 female mice (17±1 g/mouse). body weight (a) and survival rate (b) were monitored up to 8 dpi (n=6/each group). lung tissues from the infected mice were collected at 5 dpi and viral loads in the lung were measured by standard plaque assay (n=4/each group) (c). (**p < 0.01 indicates a significant difference between groups compared by student's t-test). the mice succumbed to infection and started to die by 7 dpi (showing more than 25% body weight loss) and all were died at 8 dpi. moreover, the control group mice exhibited several respiratory disease clinical signs such as decreased activity, hunched posture, huddling, and ruffled fur. mlt/ h1n1 treated mice showed increased protection and body weight with no clinical signs indicating mlt can protect the mice from lethal virus infections. moreover, influenza virus transmitted mainly through the respiratory system of mice and replicated most efficiently in lungs (bouvier and lowen, 2010) , lung titer was evaluated in pbs/h1n1 or mlt/h1n1 treated mice. mlt/h1n1 treated mice showed lower lung viral titer (log10 tcid50 1.53 ± 0.25) than the pbs/h1n1 treated mice (log 10 tcid 50 4.22 ± 0.27) at 5 dpi (fig. 7c) . these results indicate that mlt has ability to inhibit viral replication against lethal infections of influenza a viruses. animal venom and their derivatives are potential natural agents for innovative drug development against several diseases, however, many of their components and functions are fully unexplored. bv is a cocktail of natural peptides with diversity of biological activities, hence bv and its components are gaining interests to consider valuable resource for use in drug design and development. in this study, potential antiviral activities of bv against vsv-gfp virus infection were first evaluated using pre-treatment, co-incubation or post-treatment experiments ( fig. 1 ) interestingly, bv inhibited the replication of vsv-gfp in all the experiments. among the three different modes of antiviral activities of bv studied, we mainly focused on the antiviral effect of bv by the co-incubation experiments. bv mainly constitutes mlt, pla2, biologically active amines (apamin, adolapin), diverse peptides and non-peptide components (habermann, 1972; lariviere and melzack, 1996; park et al., 2004; son et al., 2007) , thus we investigated antiviral activity of bv main constituents using the co-incubation experiment. as a result, it was found that mlt the only compound that showed the similar effects with bv (data not shown). mlt constitutes almost 40-50% of dry bv and is an α-helical cationic peptide with hemolytic activity in human erythrocytes (tosteson et al., 1985) and other eukaryotic cells. it has a broad antimicrobial spectrum due to its ability to disrupt lipid cell membranes (habermann, 1972; tosteson et al., 1985) . mlt rapidly binds to human red blood cells already in sub micro-molar concentrations, induces release of hemoglobin, increase membrane permeability, and finally lysed the cells (degrado et al., 1982) . mlt disrupts cell membranes and kills gram-negative and gram-positive bacteria in micro-molar concentration range (maher and mcclean, 2006) . besides its powerful antimicrobial and lytic activity, mlt exhibits a variety of other biological effects. mlt hinder transport pumps, such as (h + + k + ) atpase (cuppoletti et al., 1989) and (na + + k + ) atpase (cuppoletti and abbott, 1990) and accelerate the permeability of cell membranes to ions. it induces inhibition and aggregation of membrane proteins (clague and cherry, 1988; mahaney and thomas, 1991) , and also mlt stimulates activity of phospholipase a2 (kuchinka and seelig, 1989) . furthermore, mlt could interfere with the activity of cellular enzyme, na+, k+ atpase, which responsible in membrane fusion process and suppresses cell fusion mediated by hsv-1 syncytial mutants (baghian and kousoulas, 1993) . matanic and castilla (2004) speculate that broad spectrum of antiviral actions might be due to ability of mlt to affect ion gradients across cell membrane. previous studies also showed that mlt display anti-viral activity against animal and plant viruses such as murine retroviruses (esser et al., 1979) , hsv-1 and hiv-1 (wachinger et al., 1992 (wachinger et al., , 1998 baghian et al., 1997) and tobacco mosaic virus (tmv) (marcos et al., 1995) , nevertheless, the mechanism of mlt activity has been not fully discovered in most cases. in this study, antiviral effect of mlt was examined against broad spectrum of viruses. mlt exhibited antiviral activity against enveloped viruses such as influenza a virus (pr8), vesicular stomatitis virus (vsv), respiratory syncytial virus (rsv), and herpes simplex virus (hsv). additionally, mlt also inhibited the replication of non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). based on our data, virus infectivity in the cells has decreased as the incubation time of the virus and mlt increased, and in the attachment or entry assay, mlt was unable to inhibit virus infection and replication (fig. 5 ). velocity sedimentation ultracentrifugation followed by immunoblotting convinced that mass of the pr8-gfp co-incubated with mlt was reduced compared to native pr8-gfp (fig. 6) . consequently, it could suggest that antiviral activity of mlt was mainly occurred due to involvement of direct interaction with the virus surfaces. this direct effect may involves to destabilize the virus structure and inactivates the viral particles (virucidal activity) which interrupts the virus infectivity. these results suggest that mlt may binds to the virus surface of the enveloped virus by surface charge interaction in virus and decrease the infectivity of the viruses (li et al., 2011) . previously, esser et al. (1979) showed that the antiviral activity of mlt has been attributed to direct lysis of viral membranes, which demonstrated to murine retroviruses. although electron microscopy or crystal structure analysis of the mlt interacting with the viral membranes has not been conducted in this study, previous information of the mlt crystal structure analysis and bio physical properties (a-helical, amphipathic, hydrophobic, and cationic) (dempsey, 1990) , it could assume that mlt may also able to interact with the phospholipid bilayer of viral envelope and cause alterations of lipid organization in the membrane as reported for hemolysis (degrado et al., 1982; terwilliger et al., 1982) or forms of ion-permeable channels in the way of the voltage gated 'pore' (tosteson et al., 1985) or micellize disc formation in the membranes which could diffuse cell contents through the holes produced (dufourc et al., 1986a (dufourc et al., , 1986b . however, the exact mechanism involves not yet identified and needs to be elucidated in future studies. moreover, one of the interesting data obtained from this study was that mlt can inactivate not only enveloped viruses, but also non-enveloped viruses such as enterovirus-71 (ev-71) and coxsackie virus (h3). however, mlt effects to which constituent of the virus and extended virucidal activity mechanism against non-enveloped viruses needs to be investigated in detail. additionally, apart from the virucidal activity of the bv, it was found that pre-treatment or post-treatment of bv can inhibit the virus replication. especially, pre-treatment experiment demonstrated that bv could induce the type i ifn (ifn-β) secretion in hek293t cells (fig. 1b) . this data suggest that bv can mediate the antiviral responses in the epithelial cells by triggering the antiviral state, playing a crucial role in inhibition of viral replication. moreover, post-treat-ment experiment of bv also showed the inhibition of vsv-gfp replication. however, mlt did not show the induction of type i ifn at 2 and 3 μg/ml (data not shown). from these results, it could assume that biological active components present in the bv other than the mlt can inhibit the specific viral replication steps of vsv. consequently, it could expect that bv or specific compounds of bv might play a role for viral replication step and various innate immune pathways that can be involved in bv anti-viral activity, especially, stimulation of type 1 ifn signaling, however, mlt has no effect on the stimulation of type 1 ifn. the detail relationship between the mechanisms of the antiviral effects of bv and active compounds present, which has not been identified yet in the bv should be investigated further. cytotoxicity of the peptide is one of the main difficulty rise during the clinical usage of therapeutic peptide, hence, cytotoxicity was evaluated for the bv and mlt and it exhibited range of cc50 (4.36-8.98 μg/ml) for tested cells (table 1) . it could suggest from the data that the antiviral activity of bv or mlt against the viruses examined, were not resulted from the cytotoxicity of the peptide on the tested cells. further, in vivo studies also showed no toxicity signs in the mouse during challenge experiments (fig. 7) and mice protected from lethal infection of influenza a virus (h1n1). this suggest that mlt can be used safely as an antiviral agent against broad spectrum of viruses with in maximum efficacy dependent on the cell type, however, its safety in concern and mechanism of action needs to be elucidated more in detail in future. in summary, this study demonstrated that bv and mlt derived from bv exhibited potent antiviral activity against various enveloped and non-enveloped viruses in vitro and influenza virus in the in vivo mouse model. moreover, its antiviral mechanism has been confirmed to involve direct interaction with the viral surface. apart from bv virucidal activity, bv can stimulate type i ifn, which subsequently could stimulate the antiviral state in the host cell and also inhibit the viral replication. taken together, these results suggest that bv or mlt has potential to become prophylactic or therapeutic agent for infectious viral diseases. md bashir uddin, byeong-hoon lee designed and executed all virus infection experiments; chamilani nikapitiya, jae-hoon kim, tae-hwan kim and hyun-cheol lee executed all cell biological experiments; choul goo kim and jong-soo lee analyzed the data. jong-soo lee and chul-joong kim designed the overall study and wrote the paper. all animal experiments were approved by the institutional animal care and use committee of bioleaders corporation (reference no. bls-absl-13-008), and were performed in accordance with the guide for the care and use of laboratory animals from the us national institutes of health. none of the authors have any financial or personal relationships with other people or organizations that could inappropriately influence or bias this study. polyphenols and disease risk 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development of sequence database for pathotype prediction and molecular epidemiological analysis therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds trypan blue exclusion test of cell viability the structure of melittin in the form i crystals and its implication for melittin's lytic and surface activities melittin lysis of red cells activation of human inflammatory cells by secreted phospholipases a2 antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression influence of amphipathic peptides on the hiv-1 production in persistently infected t lymphoma cells interferon-mediated antiviral activities of angelica tenuissima nakai and its active components comparative study of enterovirus 71 infection of human cell lines the key: cord-268565-2sg1tlrg authors: clarke, david k.; cooper, david; egan, michael a.; hendry, r. michael; parks, christopher l.; udem, stephen a. title: recombinant vesicular stomatitis virus as an hiv-1 vaccine vector date: 2006-09-15 journal: springer semin immunopathol doi: 10.1007/s00281-006-0042-3 sha: doc_id: 268565 cord_uid: 2sg1tlrg recombinant vesicular stomatitis virus (rvsv) is currently under evaluation as a human immunodeficiency virus (hiv)-1 vaccine vector. the most compelling reasons to develop rvsv as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. numerous preclinical studies with rvsv vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rvsv vaccine platform. when administered to nonhuman primates (nhps), rvsv vectors expressing hiv-1 gag and env elicited robust hiv-1-specific cellular and humoral immune responses, and animals immunized with rvsv vectors expressing simian immunodeficiency virus (siv) gag and hiv env were protected from aids after challenge with a pathogenic siv/hiv recombinant. however, results from an exploratory neurovirulence study in nhps indicated that these prototypic rvsv vectors might not be adequately attenuated for widespread use in human populations. to address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rvsv vectors, are currently under investigation. additional modifications of further attenuated rvsv vectors to upregulate expression of hiv-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation. hosts without a major role in virus spread. in contrast, there is circumstantial evidence that biting insects are a major virus reservoir and vectors of disease in animals. many vesiculoviruses have been recovered in nature from bloodsucking insects [17, 102, 107, 109] and bite transmission of vsv ind and chandipura to laboratory animals by experimentally infected mosquitoes and sandflies has been demonstrated [6, 46, 80, 103, 105, 109] . in addition, natural infection has occurred in sentinel animals exposed in areas of known virus activity [47, 104] . the hypothesis that insects are the major vsv reservoir is further supported by the demonstration of vertical transmission in experimentally infected sand flies that, in nature, would allow virus to propagate and persist in insect populations without the need of an intermediate host [103, 106, 110] . vsv infection in humans is rare, but it can occur when animal handlers and veterinarians come in close contact with infected livestock and through inadvertent exposure of laboratory personnel [24, 42] . the most common portals of infection in humans are skin and mucous membranes of the nose, mouth, and eyes, although there is also serological evidence that suggests that some vesiculoviruses may be directly transmitted to humans through insect bites [7, 12, 47] . human infection with vsv can either be asymptomatic or may lead to disease symptoms, which include myalgia, headache, and fever that resolve in 3-5 days without complications. the standard vsv particle is bullet-shaped (65×180 nm), comprising a ribonucleoprotein core surrounded by a lipid envelope that is derived from host cell plasma membrane. the virus genome is an ∼11-kilobase single strand of negative-sense rna encoding five major viral proteins (fig. 1 ). the n protein is the most abundant virus protein expressed in infected cells and associates closely with genomic rna to form viral nucleocapsid, which is the functional template for both viral transcription and replication [115] . the p and l proteins associate to form the functional viral rna polymerase that performs both transcriptase and replicase functions [21, 74, 113] . the m protein is the most abundant protein in the virus particle and has multiple functions in the infected cell, including virus budding, inhibition of host cell gene expression [2, 8, 41, 54] , and regulation of viral transcription [13, 15] . the g protein is a type i transmembrane glycoprotein that forms trimeric spike-like structures on the virus surface [40, 55, 83, 118] , and facilitates virus attachment to host cell receptors and uptake by endocytosis. at ph <6 the g protein undergoes a conformational change resulting in the fusion of viral envelope with the membrane of endosomal vesicles [30] , releasing viral nucleocapsid and viral rna polymerase into the host cell cytoplasm to initiate the replicative cycle. after engaging the unique transcription promoter at the 3′-end of the nucleocapsid template, the rna polymerase initiates mrna transcription and proceeds toward the genome 5′-end, terminating and reinitiating transcription at each gene junction, resulting in the production of five discreet viral mrnas (vmrnas) [1, 4, 39] . during this process the transcriptase complex occasionally detaches from the nucleocapsid template at intergenic junctions and must return to the 3′ promoter to reinitiate transcription, which results in a pronounced 3′ to 5′ gradient of vmrna expression (fig. 2) . this transcription gradient allows the virus to express individual proteins at optimal levels for viral replication. for example, the l gene encoding the viral rna polymerase, which is a catalytic protein needed in relatively small amounts, is distal to the 3′ transcription promoter and is transcribed with low efficiency in infected cells. conversely, the n gene encoding a major structural protein, which is required in abundance for nucleocapsid formation, is immediately adjacent to the 3′ transcription promoter and is efficiently transcribed [112] . some time after initiation of primary mrna transcription, genome replication also begins. the mechanism responsible for switching rna polymerase from a transcription to a replication mode is not fully understood, although a number of hypotheses have been proposed [3, 9, 10, 16, 73] . the nucleocapsid is also the functional template for genome replication as it is for transcription. the viral replicase initiates rna synthesis at the exact genome 3′-end and proceeds to the 5′-end, ignoring transcription stop and start signals to produce a full-length positive sense copy of the viral genome. nascent positiveand negative-sense genomic rna is encapsidated with n protein in a cooperative, coreplicational manner [10, 59] , and the resulting ribonucleprotein complex subsequently functions as a template for further genome amplification and mrna transcription. the increased availability of amplified viral genome templates for secondary transcription leads to a rapid increase in the abundance of all viral proteins needed for mature virus particle production. the virus assembly process is not understood completely, but available data indicate that viral nucleocapsid associates with regions of the plasma membrane enriched with m and g proteins. the nucleocapsid then condenses into a form that is transcriptionally inactive, and in a process that appears to be primarily driven by the m protein [36, 48] , the plasma membrane containing g protein envelops the underlying viral nucleocapsid and mature virus particles bud from the cell surface. the g protein cytoplasmic tail (ct) does not appear to have an essential role in virus budding [85] , but does enhance efficiency of the process [97, 98] . the mature virus particles contain the viral rna polymerase necessary for further propagation in susceptible cells. until recently, directed manipulation of the vsv genome was impossible due to the instability of single-stranded rna and difficulties in defining conditions that would allow recovery of infectious virus from genomic cdna. however, as a result of pioneering work carried out in a number of laboratories, a recovery process known as "virus rescue," was developed approximately 10 years ago [57, 95, 116] . the rescue procedure involves the transfection of susceptible cells with plasmids expressing the virus n, p, and l proteins and a full-length positive sense genomic rna (fig. 3) . the n, p, and l proteins associate with the nascent genomic rna to initiate virus replication and transcription, producing infectious virus progeny. the ability to rescue vsv from cloned cdna has allowed fundamental questions on virus replication to be addressed, and has also enabled the rational design and development of recombinant vsv (rvsv) as a vaccine vector [27, 34, 35, 84, 85, 88, 89, 114] . there are many aspects of vsv biology that support the development of rvsv vaccine vectors for the treatment and prevention of human disease. the virus genome is simply organized into discreet transcriptional units (tus) for expression of viral proteins, and nucleotide signal sequences fig. 1 vsv genome organization, particle structure, and morphology. a the vsv genome encodes five major viral proteins, expressed from five discreet tus. a single 3′ transcription promoter and genome replication promoter reside within the 50-nucleotide-long leader sequence. the antigenome replication promoter resides within the 59-nucleotidelong trailer sequence. consensus transcription start and stop signals at the beginning and end of each tu, respectively, result in the synthesis of five vmrnas. genome replication occurs through a positive-sense antigenome intermediate. b the vsv particle is bullet-shaped, containing a ribonucleoprotein core surrounded by a lipid envelope that is derived from host cell plasma membrane. the n protein is closely associated with genomic rna forming viral nucleocapsid. viral rna polymerase comprises a complex between the p and l proteins and is packaged inside the virus particle. g protein trimers form spike-like projections from the viral envelope that attach the virus particle to host cell receptors. the electron micrograph shows vsv particles stained with phosphotungstic acid (reprinted with permission from rose and whitt [87]) involved in the control of viral gene expression have been defined [96] . accordingly, one or more additional tus encoding foreign proteins can be added to the vsv genome and robustly expressed under control of the 3′ transcription promoter [82, 84, 85, 89] . furthermore, the abundance of foreign protein expression can be modulated depending on proximity of the extra tus relative to the 3′ transcription promoter (unpublished data) [72] . although the mutation frequency of vsv is high [18, 37] and additional foreign genes are not essential for virus replication, the consensus sequence of foreign protein open reading frames (orfs) can be maintained over many rounds of amplification [96] , ensuring preservation of target antigens during the vaccine manufacturing process. the ability of rvsv vaccine vectors to propagate vigorously in qualified continuous cell lines also facilitates industrial scale manufacture of vaccine preparations. because human infection is rare, vsv seroprevalence in the general population is very low; therefore, preexisting immunity will not likely be a factor affecting the efficacy of future rvsv vaccine vectors, as may be the case for some other proposed viral vaccine vectors [14, 61] . indeed, the existence of many different vesiculovirus serotypes may be exploited to design multiple rvsv vaccine vectors [88] for prevention and treatment of many different diseases. vsv does not cause any obvious signs of disease after parenteral administration to either livestock or nonhuman primates (nhps) [89] , and rvsv vectors are anticipated to be innocuous in humans when administered parenterally. other desirable safety features include the lack of a dna intermediate during viral replication and the cytoplasmic site of genome replication, both of which reduce the likelihood of viral nucleic acid integration into host cell dna. furthermore, vector genome stability is enhanced because it is encapsidated and cannot undergo recombination. fig. 2 the vsv mrna transcription gradient. genes proximal to the 3′ transcription promoter are transcribed more abundantly than 5′ proximal genes. the gradient of mrna transcription arises because the rna polymerase complex often detaches from the template at intergenic junctions and can only reinitiate transcription at the 3′ transcription promoter fig. 3 rescue of rvsv from genomic cdna. virus rescue is initiated by cotransfecting plasmids (step 1) encoding the full-length viral genome and transacting viral polypeptides. t7 rna polymerase, introduced into the cell by a variety of different methods, mediates rna synthesis in the cell cytoplasm during the earliest stages of rescue, producing copies of the viral genomic rna and transcripts encoding the transacting polypeptides (n, p, and l) needed to promote de novo assembly of a nucleocapsid (step 2). functional nucleocapsid subsequently serves as a template for genome replication, transcription of all vmrnas, and accumulation of viral proteins (steps 3-5) triggering ensuing events in the viral replication cycle including virus assembly (step 6) and budding (step 7) in the prototypic rvsv vectors, foreign genes were expressed from a tu inserted at position 5 in the viral genome ( fig. 4 ) between the g and l genes [49, 84, 85, 89] . this insertion site was chosen to minimally perturb the stoichiometry of viral protein expression, which is modulated by distance of each gene from the 3′ transcription promoter. because the l protein has a catalytic function, displacing the l gene by one additional tu from the 3′ promoter was viewed as a conservative genome alteration that should not adversely affect viral replication, while allowing adequate foreign protein expression to generate an immune response. this rationale was supported by in vitro growth studies showing only minimal reduction in peak virus titers for vectors containing an extra foreign gene between the g and l genes [22, 85] . furthermore, the level of foreign protein expression was sufficient to elicit robust antigen-specific immune responses after a single inoculation of mice and rabbits [82, 84, 85] . in some cases, primary immune responses could be further boosted with a second dose of homologous vaccine 1 to 2 months later [82, 85] . however, because durable neutralizing antibodies are usually elicited against the vsv surface glycoprotein after a single vaccination with replication-competent rvsv vectors, glycoprotein exchange vectors were designed to allow more effective boosting of immune responses to target antigens. the exchange vectors were generated by switching the ind serotype g gene in priming vectors with either the vsv nj or chandipura serotype g gene in boosting vectors [88] . in view of the flexibility of the rvsv vector platform and the encouraging data from preclinical studies with experimental influenza vaccines [84, 85] , rvsv vectors expressing antigens from a variety of other human pathogens were developed and tested in preclinical animal challenge models (table 1) . other vectors expressing antigens from hepatitis c virus (hcv) [22] , hantavirus [58] , and mycobacterium tuberculosis [117] were assessed in preclinical immunogenicity studies. the first nhp experiments to evaluate rvsv as a vector system for human immunodeficiency virus (hiv) were conducted by dr. john rose and colleagues at yale. in these pioneering studies, rhesus macaques were immunized with a combination of two prototypic rvsv vectors expressing a hiv-1 89.6 env gp160/vsv-g fusion polypeptide and simian immunodeficiency virus (siv) gag p55 protein. vaccine vector administration by a combination of intramuscular and intranasal routes clearly demonstrated the ability of rvsv vectors to elicit potent antigen-specific cellmediated immune responses in nhps. in addition, this study also clearly demonstrated for the first time the ability of rvsv vectors expressing env and gag proteins to provide highly significant protection from aids after an intravenous heterologous siv/hiv (shiv) 89 .6p challenge [89] . in this study, all seven macaques receiving rvsv expressing hivenv and sivgag remained disease-free after shiv 89.6p challenge for >4 years ( [89] and j. rose, personal communication). by comparison, all eight control macaques [either unimmunized or immunized with labadapted rvsv expressing influenza hemagglutinin (ha)] progressed to aids with an average time of 8 months ( [89] and j. rose, personal communication). the protection from aids in this study correlated with large differences in peak viral loads, low or undetectable viral loads at set point, and with the preservation of cd4 + t cells in the vaccinees relative to controls. this encouraging level of postchallenge vaccine efficacy suggested that rvsv vectors expressing hiv genes might be an effective aids vaccine in humans. in addition to demonstrating postchallenge efficacy in nhps, rvsv-based vaccines have other advantages that suggest they may be ideally suited for development as a worldwide prophylactic hiv-1 vaccine. most notably, rvsv-based vaccine vectors, at least in animal models, can be safely and effectively administered via a mucosal route as shown in several animal models. for example, a single intranasal inoculation with an rvsv vector expressing the influenza virus ha protein in mice [84, 85] , the herpes simplex virus (hsv) type 2 glycoprotein d in mice [69] , or a measles virus ha protein in cotton rats [94] completely protected animals against mucosal challenge with the corresponding wild-type pathogen. recently, our laboratory systematically compared the immunogenicity of rvsv vaccine vectors when given either as an intramuscular or intranasal inoculation in an effort to determine which route of vaccine administration was optimal for inducing humoral and cellular immune responses in rhesus macaques [19] . our results demonstrated that mamu-a*01 + mhc class i-positive macaques, vaccinated with a combination of studies have not yet been conducted that directly evaluate the safety of rvsv vectors when administered to humans. as mentioned previously, infection with field isolates and cell culture-adapted vsv was not associated with significant illness in humans. furthermore, in the course of numerous immunogenicity and safety studies performed at wyeth (unpublished data), more than 150 nhps were inoculated either intranasally or intramuscularly with high doses (10 7 plaque-forming units) of cell culture-adapted vsv and a variety of rvsv vectors without adverse effects. animals maintained normal blood chemistry, blood cell count, and body weights after inoculation with virus, and showed no signs of the characteristic vesicular lesions observed in nasal and lingual epithelia. although the studies described above indicated that rvsv vaccine vectors could be administered safely to humans, additional safety studies would be required before proceeding with clinical studies. replication-competent viral vaccines have historically been subjected to stringent safety testing including direct inoculation of virus into the central nervous system of susceptible nhps to measure neurovirulence (nv) potential [60, 68, 91, 99] . although the rvsv/hiv-1 vaccine vectors described above are attenuated relative to vsv field isolates, they are replica-tion-competent and subject to the same stringent safety testing used for other live attenuated vaccine viruses. for this reason, the prototypic rvsv vector and a number of closely related rvsv vectors expressing hiv-1 gag protein were tested in an exploratory nhp nv study. this study was also warranted by historical data showing that vsv field isolates and tissue culture-adapted vsv strains were neurotrophic and neurovirulent in rodents [81, 92, 111] , and that vsv serially passaged in mouse brain was found to be neurovirulent in livestock and nhps inoculated by the intracranial route [29, 70] . the nv potential of rvsv/hiv-1 vaccine vectors was tested by direct intrathalamic inoculation of cynomolgus macaques in accordance with the procedures used to assess production lots of mumps virus vaccine [65, 91] . the results of this exploratory study showed that the prototypic rvsv vector expressing hiv-1 gag was much more attenuated than cell culture-adapted wild-type vsv, but retained a level of residual nv that would likely be unacceptable to regulatory agencies (j. erik johnson et al., submitted for publication). to address the safety concerns raised by the outcome of the exploratory nhp nv study, a variety of distinctive strategies were investigated to further attenuate growth and virulence of rvsv vectors. multiple approaches were pursued because it was impossible to predict which attenuation strategy would provide an optimal balance between safety and immunogenicity. the ability to attenuate in vitro growth and virulence of rvsv by moving the n gene from the first position in the genome to downstream locations was clearly demonstrated [27, 28, 112] . the step-wise translocation of the n gene further away from the 3′ transcription promoter leads to incremental reduction in n protein expression, thereby limiting nucleocapsid formation (figs. 2 and 5). because nucleocapsid is the functional template for mrna transcription and genome replication and is part of the virion core, limiting nucleocapsid leads to a decrease in viral protein synthesis, genome amplification, and formation of mature virus particles. in addition to providing a means of attenuating rvsv vectors incrementally, another attractive feature of this attenuation strategy is the known genetic stability of n gene translocations [4, 27] , which greatly eliminates the likelihood of reversion. truncation of the ct region of g protein was also used to attenuate in vitro growth and in vivo virulence of rvsv vectors [85, 98] . the wild-type g protein ct region is 29 amino acids in length. however, when the ct was truncated to either one or nine amino acids (ct1 and ct9, respectively) ( fig. 6) , peak virus titers obtained in cell culture were reduced, and the resulting viruses were less pathogenic in mice after intranasal inoculation [85, 98] . the attenuation mechanism likely involves an impaired interaction between the viral ribonucleoprotein core and the truncated g protein cts during viral morphogenesis [66, 71, 98] , leading to a reduction in the efficiency of virus particle assembly. as seen for n gene translocation, g protein ct truncation also gives rise to virus with a very stable attenuation phenotype because replacement of deleted sequence encoding part of a protein is very improbable. the vsv m protein plays a key role in the development of virus induced cell cytopathology and cell death [2, 8, 54] . mutations in the rvsv m gene that delay and diminish cytopathic effect in infected cells were described [41] . these mutations ablate the expression of two overlapping, in-frame polypeptides that are expressed from the m protein mrna by initiation of protein synthesis at internal augs (fig. 7) . both polypeptides may have a role in the development of cell cytopathology by inhibiting nuclear export of cellular mrna and disruption of the cell cytoskeleton [23, 41, 91] . these m gene mutants are also much less pathogenic in mice (unpublished data). historically, vsv temperature-sensitive (ts) mutants were isolated either as a result of random point mutation or, more commonly, after chemical mutagenesis [26, 76, 77] . these conditional mutants usually propagate robustly at a permissive temperature and poorly or not at all at a defined nonpermissive temperature. because the temperature of nasal epithelium is typically lower than core body temperature, selective incorporation of ts mutations should produce rvsv vectors that replicate vigorously at 32-35°c but not at 37°c, and serve as vectors for intranasal step-wise translocation of the n gene further away from the 3′ transcription promoter leads to incremental attenuation of virus growth in vitro and in vivo. this method of attenuation relies on downregulation of n protein expression due to the 3′ to 5′ transcription gradient fig. 6 attenuation of rvsv by g protein truncation. truncation of the ct region of the rvsv g protein from 29 amino acids to either nine (ct9) or one (ct1) amino acids progressively attenuates virus growth in vitro and in vivo. it is believed that viral morphogenesis is adversely affected due to impaired interaction between shorter cts and underlying viral core proteins (fig. 7b) . the molecular mechanism of ts attenuation is often not clearly defined but probably involves impaired protein folding and instability at the nonpermissive temperature. in case replication-competent rvsv vectors prove to be inadequately attenuated for use in humans, propagationincompetent rvsv vectors are also being developed. these vectors are restricted to a single round of replication and are unable to spread beyond primary infected cells. one such vector has the entire g gene deleted (δg), and therefore requires g protein transcomplementation for propagation of infectious virus particles in vitro (fig. 8 ) [85] . another related vector has most of the g protein ectodomain deleted (g stem), retaining the ct region, transmembrane domain, and 42 amino acids of the membrane proximal ectodomain (fig. 8) . this vector is also propagation-defective, requiring g protein in trans for production of infectious particles in vitro. expression of the truncated form of g protein (g stem) enhances the overall yield of progeny virus particles in vitro compared to vectors with the entire g gene deleted, perhaps due to increased concentration of g protein cts at the cell plasma membrane, facilitating virus assembly and budding [86] . although propagation-defective virus offers obvious safety advantages, difficulties in providing adequate quantities of complementing g protein to allow efficient vector amplification during industrial scale manufacture are a possible limitation. the above strategies gave rise to rvsv vectors showing a range of growth attenuation in vitro and a marked reduction in pathogenicity after intranasal inoculation of mice [77, 85, 86, 114] . however, some vectors still displayed levels of nv that were almost equivalent to unaltered prototypic rvsv vector after intracranial inoculation of mice [28, 77, 114] . in addition, one of the attenuated vectors containing a g protein ct truncation that was highly attenuated in vitro and after intranasal inoculation of mice still caused moderate levels of neuropathology in the nhp nv model (j. erik johnson et al., submitted for publication). these observations indicated that the previously described attenuation strategies might not be sufficient to reduce rvsv vector nv in animal models to a level that would be acceptable to regulatory agencies. therefore, additional strategies to further attenuate rvsv vectors are being explored, including combinations of ct truncations with either n gene rearrangements or mncp mutations (fig. 9) . it was thought that these mutation combinations would increase vector attenuation beyond levels obtained using single attenuation strategies. other types of gene rearrangement, including m and g gene shuffling, singly and in combination, are also under investigation [5, 25] . specific combinations of ts mutations are also being developed in an effort to ensure tight shut-off of viral replication at the nonpermissive temperature while retaining high levels of transcriptional activity (fig. 9) . as with other attenuated rvsv vectors, plaque size and one-step growth kinetics will serve as an initial measure of replication efficiency of further attenuated combination mutants. those vectors showing significant growth attenuation in vitro will then be tested for nv in mice after intracranial inoculation. this route of inoculation was chosen because significant differences in virulence between highly attenuated vectors are often not apparent after future approaches to rvsv attenuation. combination of attenuation strategies may further extend the range of attenuated rvsv vectors to be tested to achieve an optimum balance of safety and immunogenicity. n gene shuffles may be combined with either g protein ct truncations or mncp mutations. other individual gene translocations (m and g) may, on their own and in combination with other attenuation strategies, provide an optimal attenuation phenotype. novel combinations of ts mutations may also produce rvsv vectors that can replicate well at 34-35°c, but not at core body temperature, and may be suited to intranasal vaccine delivery intranasal inoculation, but can be measured after intracranial inoculation due to the exquisite sensitivity to vsv infection by this route [38, 67, 75, 81, 114] . the resulting mortality and morbidity can be used to calculate a 50% lethal dose (ld 50 ) for each vector, and these data will then be used to define a rank order of vector attenuation. although the murine nv model is tractable and provides a very sensitive means of ranking rvsv vector nv, additional nv studies also will be carried out in ferrets and nhps to further evaluate vector safety. ferrets were used as a disease model to study influenza virus because they are naturally susceptible to infection and display many of the disease symptoms seen in humans [63, 64, 90, 93, 100] . a ferret nv model was recently developed because components of the influenza virus particle can induce significant neuropathology in nhps after intracranial inoculation in the absence of viral replication (unpublished data). because the ferret nv model might be a better indicator of nv and neurotropism potential in nhps and humans, rvsv vector safety also will be assessed after intranasal and intracranial inoculation of ferrets. in the absence of a clearly defined marker of ferret nv (ld 50 ), changes in brain histopathology profiles will be used to assess the extent of injury associated with each rvsv vector. data from murine nv and immunogenicity studies and ferret nv studies will then be used to identify the most promising candidate vectors for further nv testing in nhps. these studies will be performed as before for the prototypic rvsv vectors. for the nhp nv studies, uvinactivated rvsv will be used as a control to measure baseline pathology resulting from the injection procedure and potential injurious effects of viral components in the inoculum. these controls are important in stringent nv testing of novel replication-competent vaccine candidates because injection of even the most innocuous material directly into the brain can lead to cell necrosis around the needle track and low-level inflammatory responses throughout the cns. typically, the two major types of injury resulting from intrathalamic inoculation of replication-competent virus are inflammatory responses stimulated by the presence of foreign material in the brain, mediated by cytokines and infiltrating leukocytes, and tissue necrosis resulting from cell death caused by viral replication. more specifically, the distinction between neuronal necrosis and inflammatory responses is important because neuronal cell necrosis is thought to be an irreversible process resulting in permanent neurological deficit, while inflammatory responses typically resolve over time, usually without permanent sequelae. therefore, rvsv vectors that produce only minimal to mild inflammatory responses in the brain would be strongly favored over those causing necrotic lesions. if, as expected, some of the further attenuated rvsv vectors currently under development cause only very minimal inflammatory responses in the nhp cns after intrathalamic inoculation and retain hiv-1-specific immunogenicity, then one or more of these vectors will be considered for future clinical evaluation. as rvsv vectors are further attenuated to enhance their safety, additional measures to maintain or increase immunogenicity may be required. the relative flexibility of the rvsv vector system and its relative ease of genetic manipulation allow for potential improvements in the immunogenicity of these vectors. there are several approaches that can be exploited to increase immunogenicity, including enhancement of foreign antigen expression, reduction of antivector immunity, coexpression of cytokines or immunomodulators, in vivo targeting of dendritic cells, and heterologous prime/boost vaccination regimens. ideally, viral vectors used as vaccines can be rationally designed such that immunogenicity can be directed away from vector-specific antigens and toward the expressed foreign antigen, thereby maximizing immunogenicity of the target antigen and minimizing immunogenicity of the vector antigens. antivector immunity can limit the ability to boost responses, and may limit the use of the same vector backbone for several vaccine targets. this might be especially relevant to immunotherapeutic approaches for persistent infections such as hiv, hsv, and hcv where repetitive boosting may be required to control infection. one potential approach to shift the focus of the immune response toward the expressed foreign antigen and away from vsv antigens is to take advantage of the transcriptional gradient of vsv described earlier. the prototype rvsv vectors developed by rose et al. [89] positioned the gene expressing the foreign antigen toward the 5′-end of the viral genome. this was done to promote efficient rescue from cdna and maintain viral replicative fitness. however, due to the 3′ to 5′ transcription gradient, positioning the foreign gene toward the 5′-end of the genome reduces the expression of the foreign antigen, relative to the four vsv 3′ proximal gene products. therefore, future rvsv vectors will be designed with the foreign antigen gene at the 3′-end of the vsv genome (position 1), thereby maximizing the expression of the foreign antigen. positioning the foreign gene in the first position in the genome should also have an attenuating effect on virus growth as all viral genes will be translocated further away from the 3′ transcription promoter, reducing their expression. one potential limitation of viral vectors is preexisting immunity to the vector. in addition, the primary immune response to the vector often limits the effectiveness of subsequent booster immunizations. in both small animal models and nhps, a strong serotype-specific neutralizing antibody response is generated against the g protein of vsv after a single inoculation. the presence of these neutralizing antibodies precludes using the identical rvsv vector as a boosting vector in animals and will presumably have the same effect after vaccination in humans. to circumvent neutralizing antibodies and create vectors for boosting, rose et al. [88] developed rvsv glycoprotein exchange vectors that encode the n, p, m, and l genes of the vsv ind serotype, but have the gene expressing the vsv ind serotype g protein substituted with either the gene expressing the vsv nj serotype g protein or the gene for the g protein of the related vesiculovirus chandipura virus [88] . while these glycoprotein exchange vectors circumvent the neutralizing antibody response to the g protein, it is possible that t cell responses to the n, p, m, and l proteins might restrict virus replication and limit the ability of these vectors to boost responses. generation of a complete rvsv nj serotype vector was not attempted due to the potential for cross-reactive cytotoxic t-lymphocyte responses between ind and nj vsv serotypes. however, there are additional viruses in the vesiculovirus genus that could be used to supply g genes for future rvsv g protein exchange vectors if multiple boosting vectors are desired. attachment glycoprotein exchange of this manner does introduce a new gene into the vector, may alter the vector phenotype, and therefore careful safety testing must be conducted before advancing these vectors to clinical trials. while changing the rvsv serotype may provide the ability to boost with related vectors, more robust boosting may be possible with completely heterologous viral vectors or nonviral delivery systems such as plasmid dna, protein subunit, or bacterial vectors. studies in mice conducted with vectors expressing hiv env or gag showed that boosting rvsv-primed animals with vaccinia-based vectors produced a fivefold increase in peak hiv-specific cd8 + t cell responses relative to mice boosted with rvsv glycoprotein exchange vectors [35] . as a follow up study, ramsburg et al. [78] compared the effectiveness of a single prime-boost immunization regimen in macaques consisting of lab-adapted g protein exchange vsv vectors expressing shiv env, gag, and pol proteins to that of a protocol consisting of a lab-adapted rvsv vector prime followed by a single boost with modified vaccinia virus ankara (mva) expressing the same shiv proteins. the macaques primed with a rvsv vectors expressing shiv proteins and boosted once with a mva vector expressing shiv proteins mounted increased immune responses relative to animals boosted with vsv exchange vectors; however, due to the small numbers of macaques in each group, this difference did not achieve statistical significance. nevertheless, after intravenous shiv 89.6p challenge, mva-boosted macaques demonstrated a further 1 log 10 reduction in peak viral loads, cleared challenge virus faster, and preserved cd4 + -t cell counts to a greater extent than macaques receiving vsv g protein exchange vectors. collectively, these data suggest that while g protein exchange rvsv vectors can circumvent vsv-specific neutralizing antibody and boost vaccinespecific immune responses, vaccine-specific immunogenicity and efficacy are substantially improved by boosting with a heterologous viral vector. recently, we demonstrated that priming with a series of intramuscular injections of plasmid dna encoding interleukin-12 (il-12) and sivgag effectively enhanced the immunogenicity and postchallenge efficacy of two intranasal doses of rvsv expressing hivenv and sivgag in rhesus macaques [20] . the results demonstrated that the plasmid dna priming vaccination regimen significantly increased sivgag-specific cell-mediated and humoral immune responses, and significantly lowered viral loads post-shiv 89.6p challenge relative to macaques receiving only the rvsv vectored immunizations. the addition of the plasmid dna priming regimen also tended to increase the preservation of peripheral blood cd4 + cells and reduce the incidence of aids-like disease symptoms and death associated with shiv 89.6p infection, although this did not achieve statistical significance due to the relatively small number of macaques used per group. an analysis of immune correlates of protection after shiv 89.6p challenge revealed that the shiv-specific ifn-γ elispot responses elicited by vaccination correlated with postchallenge clinical outcome. thus, the higher shivspecific cell-mediated immune responses elicited by the dna prime/rvsv boost vaccination regimen was associated with an increased maintenance of cd4 + cells and a reduction in plasma shiv viral loads. when the rvsv hiv-1envg/ sivgag vaccines were given alone, substantially lower shiv-specific cell-mediated immune responses were elicited and the postchallenge protection afforded by immunization was reduced. once vectors based on rvsv and other platforms have demonstrated safety and immunogenicity in clinical trials, it will be important to assess their combined use in heterologous prime/boost vaccination regimens to maximize their potential. there may be additional benefit gained by creating rvsv vectors expressing heterologous viral attachment glycoproteins. in the effort to use rvsv as a vaccine vector for human diseases, many groups have either replaced the vsv g protein or coexpressed it with glycoproteins from targeted human pathogens including hantavirus [ [32, 33] . when tested in animal models, these vectors elicited immune responses directed against the expressed foreign glycoproteins. more recently, nhps immunized with rvsv vectors expressing glycoproteins from either marburg or ebola viruses were protected from disease after challenge with these pathogens [44] . moreover, it is likely that replacing the vsv g protein with filoviruses glycoproteins altered rvsv vector tissue tropism such that they could readily target dendritic cells, as filoviruses are known to do. such retargeted rvsv vectors could be designed to express additional foreign antigens from unrelated pathogens and serve as either priming or boosting vectors along with rvsv ind and nj serotype vectors. because retargeting by glycoprotein exchange can markedly alter virus phenotype and cell tropism, any new rvsv vector of this type will require extensive safety studies before clinical assessment. one advantage of using viral vectors as vaccines is that they are believed to act as their own adjuvant by stimulating the innate immune response through the binding of viral components to pathogen recognition receptors of the host cells. specifically, vsv singlestranded rna is believed to bind toll-like receptor-7 in infected plasmacytoid dendritic cells, and is essential to the initiation of a type i ifn response and other innate responses [62] . furthermore, the ribonucleoprotein complex of vsv was shown to activate cellular kinases tbk1 and ikkɛ that are involved in the production of type i ifns [101] . in spite of this adjuvant activity of vsv, and because rvsv vectors will need to be further attenuated, it may be helpful to enhance their immunogenicity through the expression of proinflammatory cytokines, or the coadministration of small molecule adjuvants and immunomodulators. several rvsv vectors have already been generated that express various cytokines. while most of these vectors have had a weak adjuvant effect, positive results from rvsv vectors expressing il-12 [52, 53] or granulocytemacrophage colony-stimulating factor (gm-csf) [79] have been reported. il-12 is a proinflammatory cytokine expressed by antigen-presenting cells as a heterodimer, and drives the development of th1-like responses. the orf for an il-12 fusion protein was inserted into a replication-restricted rvsv vector lacking the gene for the g protein (vsvδg-il12f) [52] . when vsvδg-il12f was coadministered with a weakly antigenic listerial antigen, enhanced th1-polarized t cell responses (determined by listerial-specific il-2 and ifn-γ secretion) and b cell responses (determined by listeria-specific igg titers) were generated in mice. moreover, coadministration of vsvδg-il12f and listerial antigen conferred protection from a lethal intraperitoneal listerial challenge [53] . gm-csf recruits and activates antigen-presenting cells, including macrophages and dendritic cells. an rvsv vector expressing murine gm-csf from the first position in the viral genome (vsv-gmcsf1) was shown to attenuate viral pathogenesis in mice, while retaining an immune response to the n protein of vsv [79] . memory responses to vsv n were also slightly enhanced in mice immunized with vsv-gmcsf1 compared to vsv n responses in mice immunized with rvsv expressing green fluorescent protein. it is still to be determined how coadministration of vsv-gmcsf1 along with a rvsv vector expressing a foreign antigen will modulate the response to that antigen. the development of procedures for the recovery of viruses in the order mononegavirales from genomic cdna has created the opportunity to utilize this group of viruses as vaccine vectors for the prevention and treatment of a wide range of human diseases. vsv is one of the most-studied of the nonsegmented negative strand rna viruses and is foremost among potential vaccine vectors in this group of viruses. vsv is naturally found in insects and livestock, is not considered a human pathogen, and has a very low seroprevalence in human populations. the small, simply organized viral genome can be altered to stably express one or more foreign proteins, and the resulting viral vectors can be amplified to high a titer in approved cell lines. the virus has a very broad host range and rvsv vector serotype can be changed simply by switching the surface glycoprotein with that from other vesiculovirus serotypes. vsv replicates exclusively in the cytoplasm of infected cells, does not persist in vivo, and viral nucleic acid does not integrate into host genomic dna. these desirable properties have led to the development of rvsv as a potential hiv-1 vaccine vector. signs of vsv infection in livestock are usually restricted to the occurrence of vesicular lesions around the mouth, nose, teats, and coronary bands of the hooves that typically resolve in 7-10 days without serious consequence. however, historical data showed that vsv could be lethal in mice, and was neurovirulent when injected directly into the brain of mice, livestock, and nhps. therefore, to investigate the potential neurotropism and nv of prototypic rvsv/ hiv-1 vaccine vectors, exploratory nv testing in nhps was carried out. the results of these tests indicated that the prototypic rvsv/hiv-1 vaccine vectors required further attenuation before clinical evaluation. a variety of different attenuation strategies and combinations thereof are being evaluated to define a more acceptable attenuation phenotype. a very sensitive mouse ld 50 nv model can be used to rank order rvsv vector attenuation level. vectors that cause minimal morbidity and mortality in the mouse model will then be further tested in ferret and nhp nv models to assess residual vector virulence. vectors that cause only a minimal degree of neuropathology will be targeted for clinical evaluation. balancing satisfactory levels of safety with adequate immunogenicity of further attenuated rvsv vectors will be challenging. however, upregulation of hiv-1 antigen expression, through gene positioning effects, may enhance immunogenicity and compensate for increased attenuation of virus replicative ability. coexpression of molecular adjuvants by rvsv vectors may further improve antigenicity of hiv-1 proteins. the combination of rvsv/hiv-1 vaccine vectors with other hiv-1 vaccine vectors, including plasmid dna, in heterologous prime-boost regimens also provides another promising approach to enhance hiv-1specific immune responses. sequential transcription of the genes of vesicular stomatitis virus ability of the matrix protein of vesicular stomatitis virus to suppress 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presence of measles virus-specific maternal antibodies infectious rabies viruses from cloned cdna the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus construction of a novel virus that targets hiv-1-infected cells and controls hiv-1 infection requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus morphological characterisitcs of the pathological process in the central nervous system of monkeys infected with variants of measles virus strain l-16 characterization of a human h5n1 influenza a virus isolated in 2003 activation of tbk1 and ikkvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity diseases transmitted from animals to man, 6th edn. c. c. thomas growth and transovarial transmission of chandipura virus (rhabdoviridae: vesiculovirus) in phlebotomus papatasi ecologic studies of vesicular stomatitis virus. ii. results of experimental infection in panamanian wild animals vesicular stomatitis virus, indiana serotype: multiplication in and transmission by experimentally infected phlebotomine sandflies (lutzomyia trapidoi) vesicular stomatitis virus (indiana serotype): transovarial transmission by phlebotomine sandflies isfahan virus, a new vesiculovirus infecting humans, gerbils, and sandflies in iran antigenic relationship among rhabdoviruses infecting terrestrial vertebrates natural infection of humans, animals, and phlebotomine sand flies with the alagoas serotype of vesicular stomatitis virus in colombia carajas and maraba viruses, two new vesiculoviruses isolated from phlebotomine sand flies in brazil relative neurotropism of a recombinant rhabdovirus expressing a green fluorescent envelope glycoprotein determination of molar ratios of vesicular stomatitis virus induced rna species in bhk21 cells replication of vesicular stomatitis virus defective interfering particle rna in vitro: transition from synthesis of defective interfering leader rna to synthesis of full-length defective interfering rna gene rearrangement attenuates expression and lethality of a nonsegmented negative strand rna virus regulation of rna synthesis by the genomic termini of vesicular stomatitis virus: identification of distinct sequences essential for transcription but not replication efficient recovery of infectious vesicular stomatitis virus entirely from cdna clones use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization dynamic equilibrium between vesicular stomatitis virus glycoprotein monomers and trimers in the golgi and at the cell surface acknowledgements this work was sponsored by a hiv-1 vaccine design and development team contract from the national institutes of health and national institute of allergy and infectious diseases (hvddt no1-a1-25458). key: cord-299281-5z1xminb authors: nan title: oligomerization of a membrane protein correlates with its retention in the golgi complex date: 1993-09-02 journal: j cell biol doi: nan sha: doc_id: 299281 cord_uid: 5z1xminb the first membrane-spanning domain (m1) of the m glycoprotein of avian coronavirus (formerly called e1) is sufficient to retain this protein in the cis-golgi. when the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (vsv g protein) is replaced with m1, the resulting chimera (gm1) is retained in the golgi (swift, a. m., and c. e. machamer. 1991. j. cell biol. 115:19-30). when assayed in sucrose gradients, we observed that gm1 formed a large oligomer, and that much of this oligomer was sds resistant and stayed near the top of the stacking gel of an sds-polyacrylamide gel. the unusual stability of the oligomer allowed it to be detected easily. gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the golgi complex formed sds-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. oligomerization was not detected immediately after synthesis of gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. furthermore, oligomerization did not occur under several conditions that block er to golgi transport. the lumenal domain was not required for oligomerization since another chimera (alpha m1g), where the lumenal domain of gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an sds-resistant oligomer, and was able to form hetero-oligomers with gm1 as revealed by coprecipitation experiments. sds resistance was conferred by the cytoplasmic tail of vsv g, because proteolytic digestion of the tail in microsomes containing gm1 oligomers resulted in loss of sds resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. interestingly, treatment of cells with cytochalasin d blocked formation of sds-resistant (but not sdssensitive) oligomers. our data suggest that sds-resistant oligomers form as newly synthesized molecules of gm1 arrive at the golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. the oligomerization of gm1 and other resident proteins could serve as a mechanism for their retention in the golgi complex. ization did not occur under several conditions that block er to golgi transport. the lumenal domain was not required for oligomerization since another chimera (otmlg), where the lumenal domain of gml was replaced by the o~ subunit of human chorionic gonadotropin, also formed an sds-resistant oligomer, and was able to form hetero-oligomers with gml as revealed by coprecipitation experiments. sds resistance was conferred by the cytoplasmic tail of vsv g, because proteolytic digestion of the tail in microsomes containing gml oligomers resulted in loss of sds resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. interestingly, treatment of cells with cytochalasin d blocked formation of sds-resistant (but not sds-sensitive) oligomers. our data suggest that sds-resistant oligomers form as newly synthesized molecules of gml arrive at the golgi complex and may interact (directly or indirectly) with an actinbased cytoskeletal matrix. the oligomerization of gml and other resident proteins could serve as a mechanism for their retention in the golgi complex. t he golgi complex is the site of oligosaccharide processing and sorting for proteins that are destined for delivery to secretory granules, lysosomes, the plasma membrane, and the extracellular space. the golgi-resident proteins that perform these functions must be retained in the golgi despite the large amount of lipid and protein traffic through this organelle. retention of these proteins is thought to involve recognition of positive signals that specify golgi localization. a useful model protein for studying golgi retention signals has been the m glycoprotein (formerly called el) of the avian coronavirus infectious bronchitis virus (ibv)k coronaviruses bud into the intermediate compartment or golgi complex of mammalian cells (griffiths and rottier, please address all correspondence to dr. carolyn machamer, department of cell biology and anatomy, the johns hopkins school of medicine, 725 n. wolfe street, baltimore, md 21205. dr. ann m. swifts current address is smithkline beecham pharmaceuticals, philadelphia, pa. 1992). when cdna encoding the m protein is expressed in animal cells, the protein is targeted to the cis-golgi (machamer et al., 1990) . structurally, the ibv m protein consists of a short glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxyterminal cytoplasmic domain. the first membrane-spanning domain (ml) of the m glycoprotein is sufficient to retain this protein in the golgi (machamer and rose, 1987) . furthermore, the ml domain can confer golgi localization to a wellcharacterized plasma membrane protein (the g protein of vesicular stomatitis virus, or vsv g). vsv g is a type i membrane protein which is cotranslationally inserted into the er, where it forms homotrimers. it is then rapidly transported through the golgi complex (as determined by the kinetics of n-linked oligosaccharide processing) and delivered to the plasma membrane (for review see doms et al., 1993) . when the membrane-spanning domain of vsv g is replaced with the ml domain of ibv m, the resulting chimera (gml, see fig. 1 ) is retained in the golgi complex (swift and machamer, 1991) . extensive mutagenesis suggests that at least four specific amino acids in the ml domain are critical for golgi retention of gml: asparagine 465, threonines 469 and 476, and glutamine 480 (machamer et al., 1993) . interestingly, these residues form an uncharged polar face along one side of the alpha helix predicted for ml. the polar nature of this face of the helix suggests that protein-protein interactions along this face may mediate golgi retention of gml. there are two general ways to envision the specific retention of a protein by information contained within its transmembrane domain (for review see machamer, 1991) . the first involves the recognition of this domain by a specific receptor that either blocks further movement in the exocytic pathway or retrieves escaped proteins back to the appropriate compartment. a receptor-based model also requires a mechanism for retention or recycling of the receptor itself. the second mechanism invokes changes in the tertiary or quaternary structure of the protein that are induced via the membrane-spanning domain when the protein encounters a particular microenvironment. changes in protein structure could include oligomerization or aggregation (covalent or non-covalent) with other proteins or lipids, which would prevent movement of the protein into transport vesicles. the cis-golgi may differ from the er in lipid composition and divalent cation concentration. these changes in microenvironment could conceivably trigger a conformational shift in golgi resident proteins upon their arrival in the appropriate compartment. we have been investigating the mechanisms by which ml might mediate retention of gml in the golgi complex. we observed that gml formed a large oligomer soon after synthesis when assayed by sucrose gradient centrifugation. here, we show that this oligomer is unusually stable, and that its formation correlates with arrival and retention in the golgi complex. our findings point to a possible role for oligomerization in the retention of gml. aprotinin, colchicine, carbonyl cyanide m-chlorophenylhydrazone (cccp), cytochalasin d, tpck-trypsin, and soybean trypsin inhibitor were obtained from sigma chem. co. (st. louis, mo). brefeldin a was from epieentre technologies (madison, wi). stock solutions of reagents were stored at -20°c. cos-'/and hela cells were maintained in dme with 10 and 5% fcs, respectively. cos-7 cells plated in 35-mm dishes (40-70% confluent) were transfected with an sv40-based expression vector using deae-dextran (machamer et al., 1985) . expression was analyzed at 40--48 h posttransfection. for expression using the vaccinia-t7 system, hela cells (40-70% confluent) were infected with the recombinant vaccinia virus vtf7-3 encoding t7 rna polymerase (fuerst et al., 1986 ) at a multiplicity of infection of 10-20. after adsorption for 30 min at 37°c, the inoculum was replaced with 0.75 ml of serum-free medium containing 2/~g of a vector (par2529) encoding the appropriate gene behind the t7 promoter and 5-10/~1 of the cationic lipid ~transfectace" (gibco brl, gaithersburg, md; rose et al., 1991) . expression level was varied by changing the amount of dna added per well (0.02-5 ~tg/dish). in coexpression experiments, cells were transfected with 1/~g of dna encoding each construct. expression was analyzed by metabolic labeling starting 3 h after infection. gml and related mutants were generated using the kunkel method of oligonucleotide-direeted mutagenesis as described previously (swift and machamer, 1991; machamer et al., 1993) . point mutants were named by appending the original amino acid (in single letter code) followed by the new amino acid. ,m (guan et al., 1988a) and tmr (doms et al., 1988) were obtained from jack rose (yale university). construction of ,~mlg was described previously (swift and machamer, 1991) . tgml was constructed from gml by inserting a stop codon at position 487 using site-directed mutagenesis (kunkel et al., 1987) . indirect immunofluorescence staining of transiently transfected cos-7 ceils was performed as described (machamer and rose, 1987; machamer and rose, 1988) . gml and related mutants were detected using a monoclonal anti-vsv g antibody (5/~g/ml; lefrancois and lyles, 1982) followed by texas red-conjugated, affinity-purified goat anti-mouse igg (10 /zg/ml; jackson immuno research laboratories inc., avondale, pa). cells expressing vsv g or mutant g proteins were rinsed once with pbs, starved for 15 rain in methionine-free dme, and labeled for the indicated times with 100 #ci/ml trans35s-label (icn radiochemicals, irvine, ca) or l-[35s] in vitro cell labeling mix (amersham corp., arlington heights, il) in serum-free, methionine-free dme. cells were solubilized immediately or after the appropriate chase period in serum-free dme containing a threefold excess of unlabeled methionine. cells were lysed in detergent solution (50 mm tris, ph 8.0, 1% np-40, 0.4% deoxycholate, 62.5 mm edta, and 0.13 tiu/ml aprotinin). samples were immunoprecipitated using a polyclonal anti-vsv antibody (produced by immunization of rabbits with purified vsv) and fixed staphylococcus aureus (calbiochem-behring corp., san diego, ca). after heating to 100*c for 3 rain in laemmli sample buffer containing 5 % fl-mercaptoethanol, samples were electrophoresed on 10% sds-polyacrylamide gels as described (laemmli, 1970) . marker proteins were 14c-methylated standard molecular weight markers (amersham corp.). labeled proteins were detected by fluorography (bonner and laskey, 1974) . am chimeras were radiolabeled and solubilized as above but immunoprecipitated using anti-human chorionic gonadotropin (hcg) antibody (organon teknika-cappel, west chester, pa) and electrophoresed on 12 % sds-pelyacrylamide gels. brefeldin a (5 /tg/ml final concentration) was diluted into serum-free medium from a 5 mg/ml stock in ethanol immediately before use. colehicine (25 mm) was prepared in dmso, diluted 1:10 into serum-free, methionine-free medium, and added to cells (50 ~tm final concentration) starting 1 h before metabolic labeling. cytochalasin d (0.1 m in dmso) was added to cells (100/~m final concentration) 2 h after infection and ineluded in subsequent changes of medium. an equal volume of dmso was added to control cells. in experiments involving cell treatment with cccp, ceils were starved in glucose-, methionine-, and cysteine-free medium for 15 rain before metabolic labeling in the same medium for 5 win. cccp (0.1 m stock in dmso) was added at the beginning of the chase in glucosefree medium to a final concentration of 300 #m. hela cells were transfected, metabolically labeled for 15 min, and chased for 45 rain as described above. cells were rinsed three times in pbs supplemented with 1 lnm mgci2 and 0.1 mm cac12, and then biotinylated using sulfo-nhs-biotin (pierce chem. co., rockford, il) as described (lisanti et al., 1988) . cells were then solubilized as described above and gml and related proteins immunoprecipitated. samples were eluted from the s. aureus pellets by heating to 100°c in 50/~1 10 mm "iris, 50 mm nac1, 1% sds, ph 7.4, and divided into two equal aliquots. one aliquot was kept at -20°c ("total") while the other was diluted tenfold with 10 mm tris, 50 mm naci, ph 7.4, and incubated with streptavidin-coupled agarose beads (pierce chem. co.) for 60 min at 4"c. the streptavidin beads were then pelleted and washed three times in ripa buffer. non-biotinylated proteins in the supernatant were precipitated using "i~a. samples were analyzed by sds-page followed by fluorography. oligomerization of the gml protein was analyzed by velocity gradient centrifugetion in sucrose performed essentially as described (doms et al., 1987) . continuous 5-20% (wt/wt) sucrose gradients were poured over a 60% (wt/wt) sucrose cushion (0.4 nil) in sw50.1 tubes. all solutions were in mnt (100 mm naci, 20 mm tris, 30 mm mes, ph 5.8) containing 0.1% triton x-100. hela cells expressing either vsv g or gml were metabolically labeled for 5 min and harvested (in mnt containing 1% triton x-100) either immediately or after 60 min of chase. lysates were loaded on top of the gradients and spun at 44-45,000 rpm for 15-16 h. fractions (0.35 ml) were collected from the top using a buchler auto densi-flow iic, immunoprecipitated with anti-vsv antibody, and electrophoresed to determine the location of the proteins in the gradient. for estimating the size of the oligomer, gradients (5-20% sucrose [wt/wt] in 100 mm nac1, 50 mm tris, ph 5.8, 0.1% triton x-100) were spun at 47000 rpm for 4 h. the markers used were thyroglobulin (19.3 $20,,0) and eatalase (11.3 $20,,0). microsomes were prepared after metabolic labeling and the appropriate chase period from transfected hela cells plated in 6-cm dishes. cells were rinsed in pbs and swelled in 0.8 ml swelling buffer (10 mm tris, 15 mm naci, t mm mgci2, ph 7.4) for 5 rain on ice. after scraping with a rubbet policeman, 0.25 ml swelling buffer containing 40% sucrose (wt/vol) was added, and the cells were homogenized in a i ml dounce homogenizer with 60 strokes of the a pestle. lysates were centrifuged briefly (90 s 5,000 rpm) to remove nuclei and large debris. 0.3 ml swelling buffer containing 10% sucrose (wt/vol) was added, and the lysates were centrifuged for 10 min at 100,000 rpm in a beckman tl-100 ultracentrifuge (t100.2 rotor). pellets were resuspended gently in pbs and divided into two aiiquots. tpck-trypsin (20/~g) was added to one aliquot, and both were incubated at 37"c for 30 min. proteolytic digestion was stopped by adding 20 #g of soybean trypsin inhibitor to both tubes. samples were solubilized (in detergent solution containing 0.4% sds or in mnt as noted) and processed further as described in the text and figure legends. previous studies from this laboratory showed that the first membrane span (ml) of the m glycoprotein of 1bv (previously called the e1 glycoprotein) causes the chimeric protein gml to be retained in the golgi. the sequence of ml is shown in fig. 1 a. using site directed mutagenesis, four key residues in the ml domain were shown to be critical for golgi retention of gml: asparagine (n,65), two threonines (t~ and t,76), and glutamine (q~). some substitutions at q~0 are tolerated (e.g., gmlqi'hs0) whereas other changes (e.g., gmlqi~o) result in transport of the protein to the cell surface (machamer et al., 1993) . another mutation in which two isoleucine residues were inserted near the center of the transmembrane domain (gmli,~) also disrupts retention in the golgi complex. these three chimeric proteins (gml~, gmlqlso, and gmlqh_~o) were expressed in transiently transfected cos-'/ cells and localized by indirect immunofluorescence ( fig. 1 b) . vsv g and grnlqi~o were readily detected at the plasma membrane, while gml and gmlqi-hso were detected only in the golgi region and largely colocalized with lens culinaris lectin staining, even after treatment with nocodazole or brefeldin a (machamer et al., 1993) . gmlin~ staining was less prominent at the plasma membrane than gmlqi~0, and a considerable amount of fluorescence was detected in the golgi complex. the localizations of these proteins were confirmed in hela cells and bhk cells transfected using a vaccinia-virusmediated expression system (not shown). interestingly, when we analyzed expression of these constructs in hela cells and cos-7 cells by metabolic radiolabeling and immunoprecipitation, we noticed the appearance of a radiolabeled sds-resistant species that migrated at the top of the stacking gel, in addition to products migrating at the expected molecular masses. the material at the top of the stacking gel was more prevalent in samples immunoprecipitated from hela cells ( fig. 1 c) than in those from cos-7 cells ( fig. 1 d) . this species was present only in immunoprecipitates from cells that expressed mutants with golgi-resident populations (gml, gmli,, and gmlqh4s0) and it appeared regardless of the level of expression (over a >50-fold range). although ~50% of the total radioactivity immunoprecipitated from gml-expressing hela cells migrated at the top of the stacking gel, we have not quantitated this information routinely. one problem with quantitation is that this material could include radiolabeled components other than gml; furthermore, some of the oligomer may not enter the gel at all and be lost during subsequent processing. gmli,, was previously reported to move through the golgi complex rapidly (with a half time of 35 rain in cos-7 cells; swift and machamer, 1991) . however, the kinetics of transport reflect only the population of protein that enters the separating gel. the sds-resistant protein may therefore represent a stable golgi-resident pool. the sds-resistant oligomerization did not depend on the type of cells used for expression, since the same results were found using transiently transfected bhk cells and stably transfected cho cells (not shown). because the sdsresistant material was easiest to detect in hela cells, we used these cells for subsequent experiments. the material at the top of the stacking gel could be detected after transfer to nitrocellulose and probing with anti-vsv g antibodies by immunoblotting, suggesting that it contained gml (or the related constructs; data not shown). furthermore, this large species was efficiently precipitated by several conformationsensitive anti-vsv g monoclonal antibodies that do not recognize grossly misfolded proteins (doms et ai., 1988) . we tried to solubilize the gml oligomer using a wide variety of lysis conditions and sample treatments. none of the modifications we tested, including changes in salt concentration, reducing agents, detergents, chaotropic agents (e.g., urea, guanidinium hc1), or temperature of solubilization or sample elution disrupted the sds-resistant oligomer (data not shown). a very small amount of sds-sensitive gml could be produced when the top of the stacking gel (containing the sds-resistant oligomer) was isolated after electrophoresis, placed in the well of a fresh gel, and reelectrophoresed (not shown). together, these observations suggest that sds-resistant oligomer formation is an intrinsic property of gml and mutant gml proteins that are retained in the golgi complex, but not of mutants that efficiently reach the plasma membrane. if the oligomerized protein represents a distinct intracellular pool of protein(s), it should not be susceptible to cell surface biotinylation. moreover, we predicted that an oligomer that formed nonspecifically after cell solubilization would be precipitated by streptavidin since incorporation of a few biotinylated molecules would allow precipitation of the entire oligomer. hela cells expressing gml, vsv g, or gmlin, were metabolically labeled for 15 min, and then chased for 45 min before cell-surface biotinylation with sulfo-nhsbiotin. after immunoprecipitation, an aliquot of each sample was treated with streptavidin-coupled agarose, and biotinylated and non-biotinylated proteins were recovered and analyzed by sds-page (fig. 2) . under these conditions, ,o50% of the total vsv g was biotinylated and could be recovered in the streptavidin pellet. only the mature (sialylated) form of vsv g was recovered in this fraction, suggesting that biotinylation was indeed restricted to cell surface proteins. in contrast, essentially none of the gml was precipitated by streptavidin, suggesting that this protein remained in an intracellular compartment. a small amount of sds-sensitive gmli~, was recovered in the streptavidin pellet. more importantly, no sds-resistant material was biotinylated, suggesting that the sds-resistant gmli~ constitutes an intracellular (perhaps golgi-resident) pool that is distinct from the sds-sensitive protein, and that only a portion of gml~, is transported to the plasma membrane where it can be biotinylated. this result strongly suggested that oligomer formation occurs before cell solubilization. the time course of gml oligomerization was determined. hela cells expressing gml were metabolically labeled for 5 min, chased for the indicated times, solubilized, and immunoprecipitated with anti-vsv g antibody (fig. 3) . oligofigure 2 . sds-resistant oligomers are not subject to cell surface biotinylation. ceils were metabolically radiolabeled for 15 min, and then chased for 60 rain. cell surface-specific biotinylatiun was performed at 4"c using sulfo-nhs-biotin, and the cells were solubilized and immunopreeipitated with anti-vsv antibody. the bound protein was eluted from s. aureus pellets and divided in half. one aliquot was solubilized in sample buffer (t), and the remainder was incubated with streptavidin-coupled agarose beads. the supematant (is], unbound material) was collected and tca precipitated, and the streptavidin beads (p) were washed and solubilized before sds-gel electrophoresis. merization was not detected immediately after synthesis of gml, but occurred gradually with a lag of ~10 min. the kinetics of gml oligomer formation were consistent with the time at which newly synthesized proteins arrive at the cis-golgi. to determine if oligomerization occurred in a post-er compartment, we asked whether the sdsresistant oligomer would form when er to golgi traffic was blocked by incubation at 16°c. at this temperature, newly synthesized proteins accumulate in the intermediate compartment (saraste and kuismanen, 1984) . hela cells were transfected with constructs encoding gml or vsv g, metabolically radiolabeled for 5 min, and then chased at 16 or 37°c as indicated for up to 90 min (fig. 4) . no oligomer formed when cells expressing gml were chased at 160c; however, if cells chased at 16°c were subsequently transferred to 370c, oligomer formation occurred. in separate experiments, we found that the kinetics of oligomer formation at 37°c were unaffected by the inclusion of a 160c pretreatment step (data not shown). in addition, treatment with 0.3 mm cccp during the chase period also inhibited sds-resistant oligomer formation (not shown). this treatment blocks transport of newly synthesized proteins from the er. these experiments suggested that gml oligomer formation normally occurred in a post-er compartment. however, brefeldin a pretreatment (or treatment during chase) did not block oligomer formation (not shown), although it did cause redistribution of gml to an er-like pattern (machamer et al., 1993) . to ask if gml oligomerization was an artifact generated during sds-page, we analyzed oligomer formation using velocity gradient sedimentation in sucrose (doms et al., 198% gml does not occur at 16°c. transfected hela cells were metabolically radiolabeled for 5 min, and then chased at 16 or 37°c for up to 90 rain. one set of dishes was incubated for 90 min at 16°c, then transferred to 37°c for 60 min. vsv g and related proteins were immunoprecipitated from solubilized cells as described in materials and methods. 1988 ). hela cells were transiently transfected with vsv g or gml, and then metabolically radiolabeled for 5 min and solubilized either immediately or after 60 min of chase. lysates were loaded onto 5-20% sucrose gradients, centrifuged, and analyzed as described in materials and methods. immediately after synthesis, vsv g migrated as a 4s20.~ peak (fig. 5; doms et al., 1987) . after a 60-min chase, all of the vsv g had trimerized, migrating as a discrete 8s20,~ peak. the behavior of gml in these gradients was markedly different. immediately after synthesis, gml migrated similarly to the vsv g monomer. after a 60-rain chase, however, much of the gml was found in the pellet in an sds-resistant form ( fig. 5 ; note top of lane). the timedependent shift in the mobility of the oligomer in sucrose gradients suggests that oligomers already exist in the cells at the time of lysis and immunoprecipitation. the sdssensitive material in the gml pellet fractions may represent an intermediate form of oligomer that is not yet sdsresistant. alternatively, this material could contain protein released from the sds-resistant oligomer during sds-page. in contrast to our findings with gml, misfolded mutants of vsv g that aggregate in the er are found in the pellet (and are sds-soluble) immediately after synthesis (doms et al., 1988) . using different centrifugation conditions, we estimated the average size of the gml oligomer to be approximately 22s20.~, or roughly 800 kd (data not shown). therefore, if gml (mw 66 kd) is the sole component of the oligomer, we estimate there would be ,o12 molecules per oligomer. in contrast to the chimeric gml protein, oligomers of the ibv m glycoprotein are not detected when assayed by sucrose gradient sedimentation (data not shown). this might suggest that oligomerization is not a retention mechanism for native m protein; alternatively, m oligomers might readily dissociate in detergent. the resistance of the gml oligomer to solubilization suggests a structural feature of vsv g may stabilize the complex. we asked whether the lumenal domain of vsv g, which is sufficient for trimerization (doms et al., 1988) , was required for oligomer formation. to do this, we replaced the lumenal domain ofvsv g or gml with a small soluble glycoprotein, the o~ subunit of hcg. when expressed alone, the ol subunit of hcg is secreted from cells as a monomer (guan et al., 1988b) . when hcgo~ is fused to the membrane-spanning domain and cytoplasmic tail of vsv g, the chimeric protein (c~m) is membrane-bound and transported to the cell surface (guan et al., 1988a) . when the vsv g membrane-spanning domain of c~m is replaced with the ml domain of ibv m, the resulting chimera (oemlg) is retained in the golgi complex (swift and machamer, 1991) . interestingly, in metabolically labeled hela cells expressing cxmlg, we observed the time-dependent accumulation of an sds-resistant species at the interface between the separating and stacking gels (fig. 6, arrowhead) . this species was not observed in cells expressing am. the smaller l~gure 5. sucrose gradient sedimentation of gml and vsv g. hela cells transfected with vsv g or gml were metabolically labeled for 5 rain, and then solubilized immediately or after 60 rain of chase. lysates were loaded onto 5-20% sucrose gradients and centrifuged as described in materials and methods. fractions were collected, immunoprecipitated using anti-vsv antibody, and analyzed by sds-page. after the 60-min chase, all of the gml formed a large oligomer that pelleted under these gradient conditions, and much of it was sds-resistant. size of the otmlg sds-resistant oligomer is consistent with the size difference between ctmlg and gml (28 vs 66 kd). on sucrose gradients, c~mlg solubilized 60 min after synthesis migrated as a larger species (some of which was sdsresistant) than newly synthesized oanlg. in contrast, the mobility of o~m on sucrose gradients was unaltered during the chase (data not shown). thus the vsv g lumenal domain was not required for sds-resistant oligomer formation. we used a coprecipitation assay to ask if otmlg formed hetero-oligomers with gml or other related golgi-resident proteins. hela cells were cotransfected to express both otmlg and either gml, gmlins, or vsv g. after a short metabolic labeling period (5 min), cells were solubilized either immediately or after a 60-min chase. lysates were immunoprecipitated using anti-hcg antibody. when otmlg was expressed alone, the time-dependent accumulation of sds-resistant oligomer at the gel interface (arrowhead) was observed as described above (fig. 7, lane 2) . interestingly, when otmlg was coexpressed with gml or gmlins, the size of the oligomer shifted dramatically, and now migated at the top of the stacking gel (fig. 7, lanes 4 and 8) . furthermore, some proteins appeared to be solubilized during electrophoresis, since a small amount of material migrating with authentic gml and gmlins w a s also detected in these lanes (fig. 7, arrow) . in contrast, no change in the size of the oligomer was observed when c~mlg was coexpressed with vsv g (fig. 7, lane 6 ) or with gmlqls0 (data not shown), which are transported to the plasma membrane. furthermore, when cells expressing gml and c~m (containing the vsv g membrane-spanning domain) were immunoprecipitated using anti-hcg antibody, no gml was recovered (data not shown). to test the role of the cytoplasmic tail of gml in oligomer formation, we inserted a stop codon after the first residue (arg) beyond the transmembrane domain (tgml). the corresponding tailless vsv g (tmr; doms et al., 1988) trimerizes with normal kinetics but is transported slowly from the er to the plasma membrane. when tgml was localized by figure 6 . amlg forms an sds-resistant oligomer. hela cells transfected with amlg or am were metabolically labeled for 5 rain, and then solubilized either immediately or after a 60-min chase and immunoprecipitated with anti-hcg antibody. samples were analyzed by sds-page on 15 % gels. note the time-dependent accumulation of an sds-resistant species at the interface between the separating and stacking gels (arrowhead). the increased mobility of amlg and am after 60 min of chase is due to carbohydrate trimming. indirect immunofluorescence in transiently transfected cos-7 cells, some golgi staining was observed, although a significant amount of the protein was found in the er. unlike grid, tgml did not form an sds-resistant oligomer when expressed in cos-'/or hela cells, and did not sediment as a large oligomer in sucrose gradients (although it did apparently form trimers, not shown). however, because tgml appears to exit the er very slowly (like tmr), the kinetics of oligomer formation may have been too slow to detect. because tgml did not form detectable oligomers, we asked whether preformed gml oligomers could be disrupted by proteolysis of the cytoplasmic tall. the 29-amino acid figure 7 . gml and c~mlg form hetero-oligomers. hela cells were transfected with 1 #g each of mlllg and either calf thymus dna (ct), gml, vsv g, or gml~n~. after a 5-min pulse, cells were solubilized either immediately or after a 60-min chase. aliquots of the solubilized cells were immunoprecipitated with anti-hcg antibody and elcctrophoresed on 12% sds-polyacrylamide gels. the interface between the stacking and separating gels is marked with an arrowhead, and the mobility of gml in the gel is denoted with an arrow, amig forms hetero-oligomers with gml and gmlm (but not with vsv g) that could be precipitated by anti-hcg antibody. fig. 8 a) has numerous arginines and lysines that are susceptible to proteolysis by trypsin. hela cells expressing gml or vsv g were metabolically radiolabeled for 5 min, and then chased for 0 or 60 rain, and microsomes prepared as described in materials and methods. microsomes were treated with tick-trypsin for 30 rain at 37°c, and then solubilized, immunoprecipitated with anti-vsv antibody, and analyzed on sds-polyacrylamide gels (fig. 8 b) . as size standards we used radiolabeled tgml and tmr immunoprecipitated from transfected hela cells. trypsin cleaved newly synthesized vsv g protein to a species migrating slightly larger than tmr on sds-page (fig. 8 b, compare lanes 7 and 10) . after 60 rain of chase, two proteolytic products were detected. the upper band corresponded to cleavage of the cytoplasmic tail of mature (sialylated) vsv g, as it was resistant to endoglycosidase h, whereas the lower band was sensitive to endoglycosidase h treatment (not shown). in addition, we frequently observed some residual gml or vsv g were metabolically labeled for 5 rain, and then chased for 0 or 60 rain. microst,mes were prepared as described in materials and methods and treated with 20 #g tpck-trypsin (lanes 2, 4, 7, and 9) or mock treated (lanes 1, 3, 6, and 8) for 30 min at 37"c. proteolysis was stopped by the addition of trypsin inhibitor (20/zg), the microsomes were solubilized, and gml (lanes 1-4) and vsv g 0anes 6-9) were immunoprecipitated and analyzed by sds-page. more inside-out microsomes are generated from compartments further along the secretory pathway, accounting for the loss of material recovered after trypsinization of vsv g isolated after a 60-min chase (lane 9). tgml (lane 5) and tmr (lane 10) immunoprecipitated from hela cells labeled under the same conditions are included as size standards. uncleaved mature vsv g in our experiments. since the trypsin-cleaved form of vsv g migrated more slowly than t m r (whose tail contains a single amino acid), we deduced that trypsin cleaves the vsv g tall within region a marked on fig. 8 a. the proteolytic profile generated by trypsin cleavage of gml differed from that of vsv g. trypsin treatment of figure 9 . trypsin-treated gml is still oligomerized. cells expressing vsv g (a) or gml (b) were metabolically radiolabeled for 5 rain, and then chased for 45 rain. microsomes were prepared and treated with 20/zg tick-trypsin for 30 rain at 37°c, then solubilized in mnt and centrifuged on 5-20% sucrose gradients. fractions were collected, immunoprecipitated using anti-vsv antibody, and analyzed by sds-page. newly synthesized gml yielded a single species which migrated more slowly on sds-page than the vsv g proteolyric fragment. thus, even before oligomers are formed, the tail of gml is less accessible to trypsin than that of vsv g. interestingly, when gml was digested after a 60-rain chase, there was a large reduction in the amount of sds-resistant oligomer migrating at the top of the stacking gel on sds-page (fig. 8 b, compare lanes 3 and 4) . furthermore, we saw a second, larger band in addition to the one detected immediately after the pulse label. both bands were endoglycosidase h-sensitive, indicating that the proteins they represent had not passed through the medial golgi (not shown). in more detailed kinetic experiments, the amount of this upper band increased with longer chase times, with a concomitant decrease in the amount of the lower band (not shown). based on the potential trypsin cleavage sites in the figure 10 . trypsin treatment of gml and vsv g recovered after treatment with cytochalasin d. hela cells expressing gml or vsv g were treated with 100 #m cytochalasin d starting 1 h before radiolabeling. because fewer microsomes were recovered from cytochalasin d-treated cells than from untreated ceils, treated cells were radiolabeled with twice the usual concentration of radioactivity. microsomes were prepared after a 15-min pulse and a 45-min chase, and aliquots were mock treated or treated with tpck-trypsin as described in materials and methods. gml and vsv g were immunoprecipitated using anti-vsv antibody and analyzed by sds-page. cytochalasin d blocked formation of sds-resistant oligomers and accumulation of the larger trypsin-cleaved band. cytoplasmic tail and the relative mobility of these bands compared to the vsv g proteolytic fragment and tgml, we conclude that the upper and lower bands result from trypsin cleavage within regions c and b, respectively (fig. 8 a) . the upper band may represent material generated exclusively from proteolytic digestion of sds-resistant gml. this would suggest that the last three amino acids of the cytoplasmic tail of gml are required for sds resistance of the oligomer. alternatively, trypsin may destroy another (unlabeled) component of the oligomer that is required to maintain its resistance to solubilization. trypsinization of microsomes eliminated the sds-resistant form of gml. to ask if proteolysis dissociated the oligomers as well, we subjected trypsin-treated gml-containing microsomes to sucrose gradient sedimentation. microsomes prepared from hela cells after a 5-min pulse and a 60-min chase were mock-treated or treated with tpck-trypsin (20 #g) for 30 min at 37°c. samples were diluted tenfold in mnt, and aliquots were loaded on sucrose gradients and centrifuged as described in materials and methods. gradient fractions were collected, immunoprecipitated, and analyzed by sds-page. the trypsin-treated samples are shown in fig. 9 . trypsin digestion did not cause dissociation of vsv g trimers (compare fig. 9 a with fig. 5) . significantly, the larger proteolytic product of gml migrated at the bottom of the gradient, whereas some of the smaller product pelleted and some was found throughout the gradient (fig. 9 b) . this suggested that removing part of the cytoplasmic tail of gml does not disrupt the oligomer, and further supports the possibility that digestion of sds-resistant material gives rise to the larger band. we tested the possibility of cytoskeletal involvement in sds resistance of gml by pretreating cells with drugs that disrupt microtubules or actin filaments. depolymerization of microtubules with colchicine (50/~m) had no effect on the amount of sds-resistant gml formed (not shown). interestingly, treatment with cytochalasin d (100/zm) starting 1 h before metabolic labeling blocked formation of sds-resistant gml oligomers (fig. 10, lane 3) . the amount of immunoprecipitated radiolabel migrating at the top of the stacking gel decreased from 43 % of total in untreated cells to 18 % of total in cytochalasin d-treated cells (compare fig. 10 , lanes i and 3). cytochalasin d did not block the transport of vsv g since the rate of sialylation was enhanced relative to untreated cells. on sucrose gradients, gml solubilized from cytochalasin d-treated cells migrated as an sds-sensitive oligomer (in the pellet), and vsv g trimerization was unaffected (data not shown). this observation suggests that oligomer formation and sds-resistance are separable characteristics, and furthermore implies that sds-resistance may result from interaction between gml and an actin-based matrix or cytoskeleton. because cytochalasin d blocked the formation of sdsresistant oligomers of gml, we tested whether this treatment prevented the appearance of the larger proteolytic band. hela cells expressing gml or vsv g were treated with cytochalasin d (100/zm) for 1 h before a 15-min metabolic labeling. microsomes were prepared after a 45-min chase and mock-treated or treated with 20/zg of tpck-trypsin for 30 min at 37°c, and then solubilized and immunoprecipitated using anti-vsv antibody (fig. 10) . as mentioned above, treatment with cytochalasin d enhanced the fraction ofvsv g that became sialylated during this period (compare lanes 5 and 7). we routinely observed that trypsin degraded most of the vsv g recovered from cytochalasin d-treated cells (lane 8), suggesting that the orientation or durability of microsomes was affected by treatment with this perturbant. interestingly, trypsin digestion of gml recovered from cytochalasin d-treated cells yielded predominantly the smaller proteolytic species, generated by cleavage within region b in fig. 8 a. we therefore conclude that the larger gml species does indeed represent material digested from sdsresistant oligomers, and furthermore, that the oligomers may interact with an actin-based cytoskeleton. the experiments presented here support protein oligomerization as a mechanism for retaining the chimeric protein gml in the golgi complex. an unusual feature of the gml oligomer (its resistance to solubflization by sds) allowed us to easily assay oligomer formation. the specificity of oligomerization was mediated by the ml transmembrane domain, since mutant gml proteins that were retained in the golgi formed oligomers, while mutant proteins that were released to the plasma membrane did not. this specificity was preserved in another construct, otmlg, in which the lumenal domain of vsv g was replaced with the o~ subunit of hcg. furthermore, e~mlg was able to form hetero-oligomers with gml and other golgi-resident gml mutants, but not with mutants that move to the plasma membrane. this suggested that the lumenal domain of vsv g was not required for oligomerization. while the specificity of oligomerization was dictated by the sequence of the membrane-spanning region, the cytoplasmic tail of gml was necessary for sds-resistance and may play a role in oligomer formation. when the cytoplasmic tails of preexisting oligomers were digested with trypsin, the proteolytic products were soluble in sds but continued to migrate as oligomers on sucrose gradients. based on potential trypsin cleavage sites in the cytoplasmic tail of gml and the migration of the proteolytic fragments on sds-page, it is possible that removal of the last three carboxyterminal residues of gml destroys its resistance to solubilization by sds. alternatively, trypsin digestion could remove another component of the oligomer that is required for sds resistance. interestingly, cell treatment with cytochalasin d blocked the formation of sds-resistant oligomers, suggesting that the acquisition of sds resistance may reflect association of the gml cytoplasmic tail with an actin-based cytoskeleton. we are currently constructing a series of gml proteins with truncated cytoplasmic tails to determine if these proteins are correctly targeted and form oligomers. several observations suggest that gml oligomerization normally occurs after the protein leaves the er. oligomerization of gml was not detected until ~10 min after protein synthesis, which correlates with arrival of newly synthesized proteins at the cis-golgi. oligomers were not observed when er to golgi traffic was blocked by incubation at 16°c or treatment with cccp. interestingly, sds-resistant oligomers did form in brefeldin a-treated cells, suggesting that oligomers can form in pre-golgi compartments under certain conditions. brefeldin a treatment may generate the conditions required to stimulate oligomer formation in the er. conditions which could trigger gml oligomerization might include its concentration in the membrane, the retrieval of glycolipids or glycoproteins typical of the golgi complex, or changes in ph or divalent ion concentrations. we were unable to solubilize the sds-resistant oligomer using a wide variety of harsh conditions. it is possible that some of the gml oligomer is covalently crosslinked to itself or to an actin-based structure via the vsv g tail. however, removal of as few as three amino acids from the cytoplasmic tail resulted in oligomers that were sds-sensitive. this indicates that the sds resistant phenotype (which requires the last three amino acids of gml) can be separated from oligomerization per se, which is mediated by the membranespanning domain. the transmembrane domains of many proteins have been shown to mediate dimerization (sternberg and gullick, 1990; manolios et al., 1990 ; for review see bormann and engelman, 1992) . the best example is glycophorin a, whose single transmembrane domain interacts to form homodimers (bormann et al., 1989) . this dimer is resistant to solubilization by sds at ambient temperature, although resistance is overcome by dilution or incubation at higher temperatures (furthmayr and marchesi, 1976) . dimerization ofglycophorin a is highly sequence specific and appears to involve interactions between hydrophobic residues in the transmembrane domain (lemmon et al., 1992) . the transmembrane domains of the t-cell receptor (cosson et al., 1991) and of class h major histocompatibility complex molecules also appear to mediate dimer formation (cosson and bonifacino, 1992) . in the case of major histocompatibility complex molecules, o~ and/~ chains interact to form heterodimers; dimerization appears to require a pair of oppositely charged amino acids at the lumenal edge of the apposing membrane spans. a series of glycine residues on each membrane span forms a nonpolar face which allows close packing of the transmembrane domains (cosson and bonifacino, 1992) . similarly, the o~ chain of the t-cell receptor assembles with the cd3 6 chain via interaction between charged transmembrane residues (manolios et al., 1990; cosson et al., 1991) . it is not clear how the transmembrane domain of gml molecules might interact to form a large oligomer. the large lumenal domain of gml may sterically prevent direct interaction between many transmembrane domains to form an oligomer. furthermore, the polar face of the ml domain itself could be expected to interact with at most one or two similar domains. the transmembrane domains of these proteins might interact to form small clusters (dimers or trimers) of gml, mutant gml proteins, and ~mlg. these clusters could be organized into large arrays by interactions between the heads and/or tails of proteins in different clusters. these structures might contain lipids and other endogenous golgi-resident proteins. association into such an array could prevent golgi-resident proteins from entering transport vesicles and thereby function as a mechanism for their retention. the targeting signals of other golgi resident proteins from different golgi subcompartments also reside in their transmembrane domains (aoki et al., 1992; burke et al., 1992; colley et al., 1992; munro, 1991; nilsson et al., 1991; russo et al., 1992; tang et al., 1992; teasdale et al., 1992; wont et al., 1992) , although flanking domains also contribute to retention efficiency. no obvious homologies exist between the primary sequences of these membrane-spanning domains, even in enzymes thought to be enriched in the same subcompartment. thus, the membrane composition of golgi subcompartments may be responsible for specific retention of resident proteins throughout this organelle. consistent with this, no soluble resident golgi proteins have been detected to date. nilsson et al. (1993) have proposed that the cytoplasmic tails of resident proteins in sequential golgi stacks may in-teract (perhaps via a cytoplasmic linking protein) to maintain the stacks in close apposition. the formation of gml oligomers may reflect association with such endogenous structures. the combination of overexpression and our metabolic radiolabeling conditions would not be expected to reveal any long-lived endogenous golgi-resident proteins that also reside in the oligomer. however, longer radiolabeling conditions in stable cell lines expressing gml may reveal other putative components of the oligomer. we are currently performing such experiments. golgi retention of a trans-golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring region a film detection method for tritinmlabeled proteins and nucleic acids in polyacrylamide gels synthetic peptides mimic the assembly of transmembrane glycoproteins intramembrane helix-helix association in oligomarization and transmembrane signaling the transmambrane and flanking sequences of 81,2-n-acetylglucosaminyltransferase i specify med/a/-golgi localization the signal anchor and stem regions of the ~-galactoside a2,6-sialyltransferase may each act to localize the enzyme to the golgi apparatus role of transmembrane domain interactions in the assembly of class h mhc molecules membrane protein association by potential intramembrane charge pairs role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus g protein trimers differential effects of mutations in three domains on folding, quaternary structure, and intraoellular transport of vesicular stomatitis virus g protein folding and assembly of viral membrane proteins eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase subunit structure of human erythrocyte glycophorin a cell biology of viruses that assemble along the biosynthetic pathway cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin a subunit effects of altered cy~-plasmic domains on transport of the vesicular stomatitis virus giycoprotein are transferable to other proteins rapid and efficient sitespecific mutagenesis without phenotypic selection cleavage of structural proteins during the assembly of the head of bacteriophage t4 the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus glycophorin a dimerization is driven by specific interactions between transmembrane a-helices polarized apical distribution of glycosyl-phosphatidylinositolanchored proteins in a renal epithelial cell line golgi retention signals: do membranes hold the key? a specific transmembrane domain of a coronavirus e1 giycoprotein is required for its retention in the golgi region influence of new glycosylation sites on expression of the vesicular stomatitis virus g protein at the plasma membrane a single n-linked ollgosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface the e1 glycoprotein of an avian corouavirus is targeted to the cis golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted a-helix in the transmembrane domain transmembrane helical interactions and the assembly of the t cell receptor complex sequences within and adjacent to the transmembrane segment of a-2,6-sialyltransferase specify golgi retention. embo (eur the membrane spanning domain of 8-1,4-galactosyltransferase specifies trans golgi localization overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells a new cationic liposome reagent mediating nearly quantitative transfeodon of animal cells 4-galactosyltransferase: a short nh2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for golgi retention ire-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface a sequence in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization a golgi retention signal in a membrane-spanning domain of coronavirus el protein. 3 the transmembrane domain of n-gincosaminyltransferase i contains a golgi retention signal the signal for golgi retention of bovine 81,4-galactosyltrunsferase is in the transmembrane domain the 17-residue transmembrane domain of 8-galactoside a2,6-sialyltransferase is sufficient for golgi retention key: cord-000660-tsvzg0ax authors: fensterl, volker; wetzel, jaime l.; ramachandran, srividya; ogino, tomoaki; stohlman, stephen a.; bergmann, cornelia c.; diamond, michael s.; virgin, herbert w.; sen, ganes c. title: interferon-induced ifit2/isg54 protects mice from lethal vsv neuropathogenesis date: 2012-05-17 journal: plos pathog doi: 10.1371/journal.ppat.1002712 sha: doc_id: 660 cord_uid: tsvzg0ax interferon protects mice from vesicular stomatitis virus (vsv) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (isg) mediate the antiviral effect. a prominent family of isgs is the interferon-induced with tetratricopeptide repeats (ifit) genes comprising three members in mice, ifit1/isg56, ifit2/isg54 and ifit3/isg49. intranasal infection with a low dose of vsv is not lethal to wild-type mice and all three ifit genes are induced in the central nervous system of the infected mice. we tested their potential contributions to the observed protection of wild-type mice from vsv pathogenesis, by taking advantage of the newly generated knockout mice lacking either ifit2 or ifit1. we observed that in ifit2 knockout (ifit2 (−/−)) mice, intranasal vsv infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. in contrast, wild-type and ifit1 (−/−) mice were highly protected and survived without developing such disease. however, when vsv was injected intracranially, virus replication and survival were not significantly different between wild-type and ifit2(−/−) mice. when administered intranasally, vsv entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and ifit2 (−/−) mice and induced interferon-β. however, as the infection spread to other regions of the brain, vsv titers rose several hundred folds higher in ifit2 (−/−) mice as compared to wild-type mice. this was not caused by a broadened cell tropism in the brains of ifit2 (−/−) mice, where vsv still replicated selectively in neurons. surprisingly, this advantage for vsv replication in the brains of ifit2(−/−) mice was not observed in other organs, such as lung and liver. pathogenesis by another neurotropic rna virus, encephalomyocarditis virus, was not enhanced in the brains of ifit2 (−/−) mice. our study provides a clear demonstration of tissue-, virusand isg-specific antiviral action of interferon. virus infection of mammals induces the synthesis of type i interferons (ifn), which, in turn, inhibit virus replication. the high susceptibility of type i ifn receptor knockout (ifnar 2/2 ) mice to infection by a variety of viruses [1] [2] [3] provides strong evidence for the major role of the ifn system in protecting from viral pathogenesis. in these mice, although ifn is induced by virus infection, it cannot act on target cells. similarly, in genetically altered mice that are defective in ifn production due to the absence of specific pathogen-associated pattern recognition receptors, signaling proteins or specific transcription factors, viral pathogenesis is enhanced [4] [5] [6] . although the critical importance of the ifn system in regulating viral pathogenesis is now well established, in many cases it is still unclear how ifn inhibits the replication and spread of a specific virus in vivo. in this context, activation of different components of the immune system plays a major role in controlling viral diseases that are relatively slow to develop [7] [8] [9] . in contrast, in acute infection by viruses that cause severe pathogenesis and death within a few days after infection, protection is primarily provided by the intrinsic antiviral actions of ifn-induced proteins encoded by the hundreds of ifn-stimulated genes (isgs) [10] [11] [12] , several of which often contribute to the overall effect of ifn against a given virus. our knowledge of the antiviral and the biochemical properties of individual isg products is mostly limited to a few intensively studied examples such as pkr, oas/rnase l or mx [13] . however, recent systematic investigation of the antiviral functions of the entire family of isgs has started producing exciting new information [14] . in the above context, we have been investigating the biochemical and biological functions of the members of the ifit family of isgs, which are very strongly induced by ifn. there are three members of this family of genes in mice: ifit1/isg56, ifit2/ isg54 and ifit3/isg49; all of the encoded proteins contain multiple tetratricopeptide repeats (tpr), which mediate proteinprotein and protein-rna interactions [15] . in vitro, p56 and p54, the products of ifit1 and ifit2, respectively, bind to the translation initiation factor eif3 and inhibit protein synthesis [16] . the third member, p49, the product of ifit3, does not share this property [17] . recently, it has been reported that ifit proteins form a multiprotein complex that can bind to the triphosphorylated 59 end of rnas, an rna-species produced during the replication of some, but not all, viruses [18] . in vivo, these genes are strongly induced in brains of mice infected with west nile virus (wnv) or lymphocytic choriomeningitis virus (lcmv); surprisingly, different ifit genes are differentially induced in different regions of the brain, suggesting non-redundant functions [19] . to further explore the antiviral properties of the ifit proteins, we generated ifit1 knockout (ifit1 2/2 ) mice and challenged them with different viruses. we observed that ifit1 2/2 mice were particularly susceptible to a wnv mutant that is defective in its mrna cap 29-o methylation; the mutant virus killed ifit1 2/2 mice but not the wild-type (wt) mice [20] . here, we report on the antiviral properties of the newly generated ifit2 2/2 mice; these mice, but not ifit1 2/2 mice, were highly susceptible to neuropathogenesis after intranasal infection with vesicular stomatitis virus (vsv), a negative sense, singlestranded rna rhabdovirus. vsv replication is highly sensitive to the inhibitory action of ifn and is routinely used to assay the antiviral activity of ifn in vitro [21] . as expected, ifnar 2/2 mice are highly susceptible to vsv pathogenesis and the same is true for mice that specifically lack expression of ifnar on the cells of their central nervous system (cns) [1] . in spite of these observations, little is known about how ifn inhibits vsv replication in vivo. our new results indicate that in the brain, but not in other organs, ifit2 is a major mediator of ifn's protective effect against vsv. in contrast, ifit2 could not protect mice from neuropathogenesis caused by encephalomyocarditis virus (emcv), a picornavirus. thus, we have uncovered a virusspecific, tissue-specific and isg-specific antiviral effect of the ifn system. generation of ifit2/isg54 and ifit1/isg56 knockout mice ifit2 gene knockout (ifit2 2/2 ) mice were generated by deleting the entire protein-encoding region of the gene, which was achieved by flanking exons 2 and 3 with frt recombinase sites in c57bl/6 embryonic stem cells and excising the flanked region with flp recombinase ( figure 1a ). ifit2 2/2 mice were bred to homozygosity ( figure 1b) , and deficiency for induced expression of ifit2 protein was confirmed in lysates of ifn-b-treated primary murine embryonic fibroblasts (mef) ( figure 1c ). mice deficient for ifit1 (ifit1 2/2 ) were derived from c57bl/6 embryonic stem cells lacking the entire ifit1 coding region ( figure 1a ). genotypic homozygosity of the ifit1 2/2 mice and deficiency for ifit1 protein induction were confirmed ( figure 1b and 1c ). both knockout mouse lines were healthy and fertile. moreover, deletion of one gene within the ifit locus did not alter the pattern of induction of other adjacent gene family members, as compared to wild-type (wt) mice ( figure 1c ). to determine the impact of ifit2 on the outcome of viral infections in vivo, we compared susceptibilities of ifit2 2/2 and wt mice to vsv infection, using ifnar 2/2 mice as positive controls of enhanced susceptibility. virus was administered at a low dose [4610 2 plaque forming units (pfu)], intranasally, reflecting a natural route of infection for vsv [22] . as seen previously, 100% of ifnar 2/2 mice rapidly succumbed to vsv infection within 2 days ( figure 2a , and [1] ), after suffering symptoms of lethargy. on the other hand, 79% of wt mice survived, the remaining 21% succumbed to vsv, and this occurred later, at 7-10 days post infection (d.p.i.). in contrast, 100% of ifit2 2/2 mice died by 7 d.p.i. (figure 2a ), with most succumbing by 6 d.p.i.; thus, we observed uniform and more rapidly occurring death of ifit2 2/2 compared to wt mice after vsv infection. within 24 h before death, both wt and ifit2 2/2 mice developed neurological signs including ataxia, hind limb paralysis, and hyper-excitability. ifit2 +/2 mice displayed an intermediate survival curve, demonstrating a gene dosage effect ( figure 2b ). next, the role of a related gene, ifit1, in vsv pathogenesis was evaluated by infecting ifit1 2/2 mice. unlike the results observed with ifit2 2/2 mice, no statistically significant increase in mortality was observed in ifit1 2/2 mice ( figure 2b , 21% death for wt versus 36% for ifit1 2/2 , respectively; p.0.25). consistent with this, survival kinetics of ifit1 2/2 and wt mice were similar. increasing the virus dose by 10,000-fold (to 4610 6 pfu) did not appreciably change the survival curves of wt, ifit1 2/2 , or ifit2 2/2 mice ( figure 2c ). these results demonstrate functional differences between the two closely related proteins encoded by ifit1 and ifit2. the virus-specificity of the antiviral action of ifit2 was evaluated by infecting ifit2 2/2 mice with emcv, an unrelated neurovirulent positive-strand rna virus of the picornavirus family ( figure 2d ). ifnar 2/2 mice were highly susceptible to emcv infection with all mice succumbing within 2 d.p.i.; in contrast, wt mice died with a slower kinetics and at a rate of only 80%. notably, ifit2 2/2 mice behaved similarly to the wt mice, without enhanced or accelerated mortality ( figure 2d ). the same conclusion was true for a lower dose of emcv ( figure s1 ). the survival pattern of emcv-infected ifit1 2/2 mice also was similar to that of the wt mice ( figure 2d ). mice of all genotypes either succumbed after developing neurological symptoms, mainly hind limb paralysis, or survived without symptoms. these results demonstrate that the antiviral action of ifit2 is both virus-and ifitspecific. the uniform penetrance of neuropathogenesis and lethality of vsv-infected ifit2 2/2 mice, even at a low virus dose, prompted us to examine viral spread along its route from the nasal cavity into the cns ( figure 3a ). after intranasal administration, vsv infects in mammals, the first line of defense against virus infection is the interferon system. viruses induce synthesis of interferon in the infected cells and its secretion to circulation. interferon acts upon the as yet uninfected cells and protects them from oncoming infection by inducing the synthesis of hundreds of new proteins, many of which interfere with virus replication. vesicular stomatitis virus (vsv), a virus similar to rabies virus, is very sensitive to interferon but it is not known which interferon-induced protein inhibits its replication. here, we have identified a single interferon-induced protein as the protector of mice from death by vsv infection. knocking out the gene encoding this protein, ifit2, made mice very vulnerable to neuropathogenesis caused by vsv infection; a related protein, ifit1, did not share this property. moreover, ifit2 failed to protect mice from another neurotropic virus, encephalomyocarditis virus, nor was it necessary for protecting organs other than brain from infection by vsv. our observation that a single ifn-induced protein protects a specific organ from infection by a specific virus revealed an unexpected degree of specificity of the antiviral action of ifn. shared wt mice (n = number of animals used). c, survival of ifit2 2/2 , ifit1 2/2 and wt mice after intranasal infection with a higher dose of vsv (4610 6 pfu). d, survival of ifit2 2/2 , ifit1 2/2 , ifnar 2/2 and wt mice after infection with 5610 2 pfu of emcv. in a-d, data are cumulative from at least two independent experiments (exceptions: figure 2b , ifit2 +/2 mice and figure 2d , ifit1 2/2 mice infected in a single experiment). statistical significance of survival differences relative to wt mice is indicated by p-values; n.s., not significant; i.n., intranasal. doi:10.1371/journal.ppat.1002712.g002 the nasal epithelia including olfactory sensor neurons, which project to the outer layer of the olfactory bulbs (ob) [23] . this represents the entry step into the cns, which we examined by immunostaining of ob sections. in wt mice, vsv p protein was detected exclusively within the glomeruli of the ob at 2 d.p.i. ( figure 3b , upper right panel and [1] ), whereas in ifnar 2/2 mice, vsv antigen had spread into deeper layers of the ob ( figure 3b , lower left panel). in ifit2 2/2 mice ob, viral antigen was restricted to the glomeruli, as seen in wt mice ( figure 3b , lower right panel). this similar pattern of viral antigen expression between wt and ifit2 2/2 mice was reflected in the equivalent levels of viral rna in ob at 2 d.p.i. ( figure 3c ). in contrast, ,10 times more vsv rna was present in ob of ifnar 2/2 mice ( figure 3c , right panel, p,0.05). a comparison of the infectious viral burden between wt and ifit2 2/2 mice in the ob confirmed these findings: at 2 d.p.i., ,10 6 pfu/g of vsv was present in both wt and ifit2 2/2 mice ( figure 3d , p = 1.0). however, later in the course of infection, by day 6, viral ob titers in ifit2 2/2 mice were not significantly changed, whereas in wt mice average titers of infectious vsv as well as viral rna levels had decreased by ,10-fold ( figure 3c and d, both p,0.05). these results suggest that vsv initially enters and replicates with similar efficiency in both wt and ifit2 2/2 ob before spreading into the rest of the brain. the efficiency of vsv replication in the brain, excluding the ob, was examined by quantifying infectious vsv as well as viral rna. early after infection, at 2 d.p.i., virus titers in brains were low (,10 4 to 10 5 pfu/g) and roughly equivalent in wt and ifit2 2/2 mice ( figure 4a , p.0.25). similarly, viral rna levels at the same figure 3 . ifit2 does not inhibit vsv entry and replication in olfactory bulbs. a, schematic entry route of vsv into the central nervous system of wt mice after intranasal infection, and vsv spread within brain, as reported in the literature. ob, olfactory bulbs; cx, cortex; mb, midbrain; cb, cerebellum; bs, brain stem; sc, spinal cord. b, vsv p protein in ob of vsv-infected wt, ifit2 2/2 and ifnar 2/2 mice at 2 d.p.i., detected by immunohistofluorescence. c, vsv rna levels in ob of uninfected or vsv-infected wt, ifit2 2/2 and ifnar 2/2 mice at 1, 2 or 6 d.p.i., plotted as mean+sd on log scale; nd, none detected. d, infectious vsv titers in wt and ifit2 2/2 ob at 2 and 6 d.p.i.; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. in c and d, n = 4-8 mice per infected group accumulated from three independent experiments; in b, n = 2 mice from two independent experiments. all infections were 4610 2 pfu of vsv administered intranasally. asterisks indicate statistical significance: ** p = 0.006, * p,0.05; n.s.: not significant. doi:10.1371/journal.ppat.1002712.g003 time were low and comparable between wt and ifit2 2/2 ( figure 4b , p.0.25). however, at the same time, levels of vsv rna (380-fold, p,0.05) were much higher in the brains of ifnar 2/2 mice ( figure 4b , right panel). later in the course of infection (6 d.p.i.), brains of wt mice accumulated only ,5-fold more infectious vsv, with occasional clearance of the virus. in contrast, we detected markedly higher vsv titers in the brains of ifit2 2/2 mice (,350-fold higher compared to wt mice, p = 0.0009), reaching ,10 8 pfu/g ( figure 4a ); the high virus load likely caused the pronounced lethality. differences in viral rna levels in brains of wt and ifit2 2/2 mice at 6 d.p.i. correlated well with levels of infectious vsv ( figure 4b ). to determine whether ifit2 selectively restricts replication of vsv in particular regions of the brain, we measured viral rna levels in cortex, midbrain, cerebellum and brain stem at 6 d.p.i. in wt mice, vsv rna was present prominently in the cortex, midbrain and brainstem, but not in the cerebellum ( figure 4c ), which is consistent with published results [24] . however, in ifit2 2/2 mice, viral rna was 200-fold or more (p,0.05) abundant in all regions of the brain examined, including the cerebellum. the increase of vsv replication in ifit2 2/2 brains was not due to a broadened cell tropism of the virus; immunostaining for viral p protein showed exclusive localization to neurons and not other cell types, such as astrocytes ( figure 4d ). from the above observations, we conclude that after intranasal infection by vsv, ifit2 protects mice from neuropathogenesis by suppressing replication or spread of the virus in brain neurons. ifit2 and ifit1 are induced in vsv-infected regions of ob and brain the protective effect of type i ifn signaling and in particular, ifit2, against vsv neuropathogenesis prompted us to confirm its expression in ob and brain of wt mice, and whether it was induced in a type i ifn-dependent manner. in wt ob, ifit2, ifit1, and ifn-b mrna was induced strongly by 2 d.p.i., and ifit2 and ifit1 rna remained abundant until day 6 d.p.i. (figure 5a ). the induction of these genes was dependent on type i ifn receptor in ob as well as in brain ( figure 5b and 5e, and data not shown). ifit2 suppresses vsv replication in the brain after intranasal infection. a, infectious vsv titers in wt and ifit2 2/2 brains at 2 and 6 days after intranasal infection, plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. b, vsv rna levels in brains of uninfected or vsv-infected wt, ifit2 2/2 and ifnar 2/2 mice at 2 or 6 d.p.i., plotted as mean+sd on log scale. c, vsv rna levels in different regions of the brains of uninfected or vsv-infected wt and ifit2 2/2 mice at 6 d.p.i., plotted as mean+sd on log scale. d, vsv p protein in midbrain neurons of ifit2 2/2 mice at 6 d.p.i.; detection by immunohistofluorescence-labeling of vsv-p (red) and neuron (neun) or astrocyte (gfap) markers (green); in a and b: n = 4-8 mice per infected group accumulated from three independent experiments; in c: n = 4 mice per infected group; in d: n = 2 mice per infected group; all infections in a-d were intranasal with 4610 2 pfu of vsv. nd, none detected. brains in a and b were separated from obs assayed in figure 3d and 3c, respectively. asterisks indicate statistical significance: *** p#0.0009; n.s.: not significant. doi:10.1371/journal.ppat.1002712.g004 furthermore, expression of ifit2 mrna in wt ob coincided with the presence of detectable levels of the encoded ifit2 protein ( = p54) at 2 d.p.i. and 6 d.p.i., as seen by immunohistochemistry ( figure 5c , and data not shown). ifit2 protein staining was observed in vsv-infected cells within ob glomeruli as well as in surrounding and distant viral antigen-free cells, consistent with a remote ifn-dependent induction of ifit2 expression ( figure 5c , arrowheads in magnified images of right panel). ifit1 and ifn-b mrnas were induced as strongly in ob of ifit22/2 as in wt mice, which correlated well with similar abundance of vsv rna in wt and ifit2 2/2 ob (figure 5a compared to figure 3c ). in brains, at 6 d.p.i., in contrast to ob, induction of ifit1 and ifn-b mrnas was considerably stronger in ifit2 2/2 mice compared to wt mice ( figure 5d , 5-fold and 27-fold, respectively, both p,0.005). the enhanced gene induction in vsv-infected ifit2 2/2 mice was not restricted to specific regions of the brain ( figure s2 ). enhanced cellular gene expression also was observed for several virusinduced cytokine and chemokine genes, as measured by quantitative rt-pcr ( figure s3a ). gene expression profiling of brain tissue at day 6 d.p.i., using microarray analysis, revealed that many other genes, including isgs, were also more strongly induced (table s1 ). these results demonstrated that enhanced virus replication in the brains of ifit2 2/2 mice led to enhanced type i ifn, other cytokines and isg induction, which nevertheless failed to restrict vsv replication in the absence of ifit2. wt mice are as susceptible as ifit2 2/2 mice to intracranial vsv infection our results from intranasal vsv infection indicated that ifit2 induction in the brain was mediated by type i ifn that was, in all likelihood, produced by infected cells in the ob ( figure 5a ). virus replication and resultant ifn induction at 2 d.p.i. were similar in the obs of wt and ifit2 2/2 mice (figs. 3c, 3d and 5a); presumably, the newly produced ifn diffused into the rest of the brain and induced local ifit2 expression in the wt mouse brains, prior to the arrival of the infectious virus. if this were the case, one would anticipate that direct infection of the brain, without prior action of ifn produced in infected ob, would minimize the difference between the phenotypes of wt and ifit2 2/2 mice. to test this idea, we injected a very low dose (10 pfu) of vsv intracranially. as hypothesized, wt and ifit2 2/2 mice were now equally susceptible; almost all mice died by 3 d.p.i. even at this low dose ( figure 6a ) and there were equally high virus titers and viral rna levels in the brains of mice of both genotypes ( figure 6b and 6c). concomitant with virus replication, there was similar induction of ifit1 and ifn-b ( figure 6c ) and other cytokines and chemokines ( figure s3b ). these results indicate that in the absence of prior induction of ifit2 by ifn, brain neurons are highly susceptible to vsv infection. unlike the brain, other organs of ifit2 2/2 mice are not more susceptible to intranasal vsv infection ifnar 2/2 mice succumbed within two days after vsv infection without accumulating very high vsv rna levels in the brain ( figure 4b ). these mice did not develop cns-related signs of disease, but showed severe lethargy before death, suggesting that death was due to efficient replication of the virus in peripheral organs, due to the absence of an otherwise effective type i ifn-mediated antiviral protection of the same organs in wt mice. to test this, we assessed the kinetics of vsv accumulation in brains, livers and lungs of wt, ifnar 2/2 and ifit2 2/2 mice (figure 7) . at 2 d.p.i., vsv titers were very high in the liver of ifnar 2/2 mice, reaching 10 9 pfu/g ( figure 7a ). in contrast, no or little infectious virus was detected in the liver of wt mice at 2 or 6 d.p.i., indicating efficient ifn-dependent suppression of vsv replication; intriguingly, this was also observed in ifit2 2/2 mice, demonstrating that ifit2 did not mediate the anti-vsv effects of type i ifn in the liver. in lungs, which directly received a part of the virus inoculum from intranasal inhalation of vsv, the virus also replicated efficiently in ifnar 2/2 mice, reaching 10 8 pfu/g before death ( figure 7b ). in comparison, lungs of wt and ifit2 2/2 mice exhibited much lower levels of vsv at 2 and 4 d.p.i. (3,000 to 10,000-fold lower for wt and ifit2 2/2 compared to ifnar 2/2 mice, all p,0.05). by days 5 and 6 d.p.i., the virus was cleared from the lungs of a subset of wt and ifit2 2/2 mice. in contrast, in brains from the same animals, 10 to 100-fold higher average titers (p,0.05) of vsv accumulated in ifit2 2/2 compared to wt mice at all time points between 2 and 6 d.p.i. ( figure 7c ). as expected, in wt mice, both ifit1 and ifit2 were induced not only in brains ( figure 5d ), but also in livers ( figure 7d ) and lungs ( figure 7e ); ifn-b was also induced in lungs, but not livers. ifit1, ifit2 and ifn-b mrnas were also induced in the brains of emcv-infected wt mice ( figure s3c ). these findings demonstrate an unexpected brain-restricted and virus-restricted function of ifit2 in the context of the type i ifn-mediated antiviral response to vsv infection. they also indicate that in ifit2 2/2 mice, other isgs, which presumably protect the peripheral organs of vsv-infected wt mice, are either not induced in neurons or insufficient to protect them. ifns are defined by their antiviral activities. they inhibit the replication of many, if not all, viruses mostly by direct inhibition of replication in the infected cells but also by promoting the ability of immune cells to recognize and eliminate the virus-infected cells [25] . the direct effects are mediated by isgs, which number in the hundreds, and different isgs are thought to have more potent antiviral activities toward different families of viruses [13] . however, in most cases, it is not known which isg inhibits the replication of a given virus; the rare exception is the mx-mediated inhibition of influenza viruses, the underlying effect which allowed for the discovery of ifns [26] . the task of connecting a specific ifn-induced protein to a specific antiviral action is compounded by the fact that often several ifn-induced proteins act in concert to inhibit the same virus at different stages of its life cycle. moreover, a specific ifn-induced protein may be more relevant for inhibiting a virus in one specific cell-type than another. recent systematic investigation of the specific antiviral effects of different isgs has started providing significant insight into this problem [14] . such findings are complemented by the analyses of the spectra of the antiviral effects of a specific isg or a family of isgs [27] . we have undertaken an investigation of the ifit family of mouse isgs. the corresponding human proteins are known to have antiviral activities against human papillomavirus (hpv) and hepatitis c virus (hcv), neither of which replicate in mouse cells. the anti-hpv activity of human ifit1 ( = p56) has been attributed to its ability to bind hpv e1 protein and to inhibit its helicase activity, which is essential for hpv dna replication [28, 29] . the antiviral effect on hcv, on the other hand, is manifested at the level of inhibiting viral protein synthesis as a consequence of the ability of ifit1 to bind the translation initiation factor eif3 and inhibit its various actions in translation initiation [30] . it has been reported recently that the ifit1 protein can form a complex and bind to rnas with triphosphorylated 59 ends, presumably providing another means to inhibit specific viruses that produce such rnas [18] . the ifit genes are clustered at a single locus in both human and mouse. in the latter species, two alleles of ifit3 genes are flanked on two sides by one allele of ifit2 and one allele of ifit1 [15] . to identify their physiological functions, we have separately deleted the entire coding regions of ifit1 or ifit2 genes. the ifit1 2/2 mice exhibited an interesting phenotype in allowing the replication of and resultant pathogenesis by a wnv mutant, which failed to replicate in wt mice [20] . because this mutant is defective in 29-o methylation of the cap structure of viral mrnas, its rescue in the ifit1 2/2 mouse indicates that this antiviral protein recognizes the 59 ends of mrnas, a conclusion that is consistent with the observation that, in vitro, it can bind to rnas having specific structures at the 59 ends [18] . it remains to be seen whether the proposed property of ifit proteins to recognize 59 ends of rna is connected in any way to their ability to inhibit the functions of eif3 [16] , which participates in several steps of translation initiation taking place at or near the 59 ends of mrnas. replication of vsv is highly sensitive to the antiviral activity of ifns, and vsv is widely used to determine the specific activities of ifn preparations quantitatively [21] . in spite of this strong connection, it is unclear how ifn inhibits vsv replication. an early report indicated that viral primary transcription is inhibited by ifn, but it is not known which ifn-induced protein mediates this inhibition [31] . the observed sensitivity of vsv replication in vitro is reflected in vivo. ifnar 2/2 mice are extremely susceptible to vsv infection; they rapidly die within 2 days after infection and the virus replicates to very high titers in many organs of the infected mice. the extreme sensitivity of ifnar 2/2 mice to vsv infection suggests that type i ifn provides the majority, if not all, of the protective innate immune defense. eventually, protection may be facilitated by immune cell-mediated antiviral actions, but this is a slow process that does not appear to function before 6-10 days post-infection [32, 33] . thus, it is likely that one or more isgs directly inhibit vsv replication in vivo. in this context, it has been reported that mice lacking pkr, a well-studied isg, display higher susceptibility to vsv pathogenesis [34] . however, detailed investigation of the underlying mechanism revealed that pkr did not execute ifn's antiviral action; rather, it was required for efficient induction of ifn-a/b in the infected mice [35] . in vivo vsv-infection induces ifn synthesis in many cell types, using either the cytoplasmic rig-i pathway or the endosomal tlr7 pathway [4, 36] ; however, it is unclear how pkr aids this process. our results show that ifit2 2/2 mice are highly susceptible to intranasal vsv infection and the effect is gene dosage-dependent: ifit2 +/2 mice had an intermediate susceptibility phenotype. infected ifit2 2/2 mice displayed symptoms of severe neuropathogenesis late after vsv infection accompanied by efficient replication of the virus in many regions of the brain. however, virus replication was restricted to neurons and did not spread to other types of cells in the brain, such as astrocytes. our results are consistent with the hypothesis that prior, ifn-induced, ifit2 expression in the brain restricts vsv replication. supporting genetic evidence for the requirement of ifn action is provided by the high susceptibility of the ifnar 2/2 mice, which possess the functional ifit2 gene but ifit2 is not induced by vsv infection because these mice cannot respond to type i ifn. additional evidence comes from a previous study using brain-specific ifnar 2/2 mice, which displayed a pattern of susceptibility to intranasal vsv infection similar to that of our ifit2 2/2 mice [1] . in our experimental system, the source of the ifn production was figure 5c ). ifit2 was also induced at this time in the rest of the brain, without any induction of ifn mrna ( figure 5d ) suggesting that the source of ifn was the ob. in accord with the well-established concept of ifn action, pre-induction of ifit2 in neurons, before the onset of infection, was essential for the antiviral effect. in comparison, induction of ifn and ifit2 that was concomitant with vsv infection failed to have an appreciable antiviral effect, as manifested by robust virus replication at directly infected sites, such as the obs of wt mice infected intranasally ( figure 3d ) or the brain of wt mice infected intracranially ( figure 6b ). high mortality of the infected mice correlated with high virus titers in the brains of intranasally infected ifit2 2/2 mice or intracranially infected wt and ifit2 2/2 mice. in the intranasally infected ifit2 2/2 mice, death was not preceded by widespread apoptosis in the brain ( figure s4 ). however, as expected with high viral loads, ifn and other cytokines and chemokines were strongly induced ( figures 5d, s2 and s3a) ; consequently, many isgs, except ifit2, were also induced (table s1) . pre-induced ifit2 prevents efficient vsv replication in the brain, most probably by blocking one or more essential step of the viral life cycle including viral entry, uncoating, primary transcription, viral protein synthesis, rna replication, virion assembly or egress. it also might block trans-synaptic spread of the virus, although unlike another rhabdovirus, rabies virus, vsv is not known to depend on transit from neuron to neuron. in this context, it is important to note the observations made by iannacone et al. [37] using a footpad vsv infection model. they concluded that type i ifn, produced by infected macrophages and plasmacytoid dendritic cells in infected mice, blocked infection of peripheral neurons resulting in lowered infection of the cns and prevention of neuropathogenesis. it is worth noting that in our studies, the absence of ifit2 did not affect ifn induction by vsv (figures 5a and 6c ). further investigation of the biochemical mechanism behind the observed in vivo effect of ifit2 2/2 is hampered by the absence of a suitable cell culture model of the phenomenon. for example, ifit2 was not required for mediating the anti-vsv effect of ifn in mouse embryonic fibroblasts ( figure s5 ), in primary fetal neurons or in ifit2-ablated neuroblastoma cells (data not shown), results that are not surprising given the strong tissuespecificity of ifit2 action observed in vivo (figure 7) . specific rnabinding properties of ifit proteins have been recently reported [18] . following this lead, we examined the rna-binding properties of recombinant murine ifit1 and ifit2 using vsv leader rna as the probe in an electrophoretic mobility shift assay: ifit1 bound rna with a 59-ppp end but not with a 59-oh end; however, ifit2 bound neither ( figure s6 ). to obtain meaningful leads, future investigation of this kind may require using brain extracts from infected mice to detect protein-viral rna complexes that may contain ifit2 along with adult neuronspecific proteins. our results revealed several layers of specificity of ifn action, some of which were not anticipated. first, compared to ifit2 2/2 mice, ifit1 2/2 mice were much less susceptible to intranasal vsv infection; this was true for both low and high doses of virus. this finding was surprising in view of a recent report on vsv susceptibility of ifit1 2/2 mice [18] and the observation that ifit1, but not ifit2, could bind vsv leader rna in vitro ( figure s6) . the above results demonstrate that different ifit proteins have nonredundant functions in vivo. the second layer of specificity was directed toward the nature of the infecting virus. although both vsv and emcv caused neuroinvasive disease, induced ifn-b, ifit1 and ifit2 in the brain and type i ifn action was required for protection against both viruses, ifit2 was critical only for protection against vsv; the absence of either ifit1 or ifit2 did not exacerbate susceptibility to emcv. the third layer of specificity was revealed by the organ-specific action of ifit2. in the complete absence of type i ifn action in the ifnar 2/2 mice, intranasally infected vsv replicated vigorously not only in brains, but also in livers and lungs ( figure 7a-c) . in contrast, in ifit2 2/2 mice, efficient vsv replication was restricted to the brain suggesting that ifit2 does not act as an anti-vsv isg in the liver or the lung because its absence did not impact virus titers, even though ifit2 was induced in these organs of infected wt mice ( figure 7d and 7e) . the efficient vsv replication in livers and lungs of ifnar 2/2 mice, but not wt and ifit2 2/2 mice, indicates that other isgs must have anti-vsv effects in those organs. further investigation is needed to determine the basis of neuronal specificity of ifit2 action and the identities of other isgs that inhibit vsv replication in other organs. all animal experiments were performed in strict accordance with all provisions of the animal welfare act, the guide for the care and use of laboratory animals, and the phs policy on humane care and use of laboratory animals. the protocol was approved by the cleveland clinic institutional animal care and use committee (iacuc), phs assurance number a3047-01. all experimental manipulations or intranasal instillations of mice were performed under anesthesia induced by pentobarbital sodium or isofluorane, respectively, and all efforts were made to minimize suffering. all mice used were of c57bl/6 background and of both sexes; ifit2 2/2 mice were custom-generated by taconic farms, inc. by flanking exons 2 and 3 of ifit2, encompassing the complete protein-encoding region, with frt sites in c57bl/6 embryonic stem (es) cells, and deleting the flanked region by transfection of flp recombinase. es cell clones were injected into bl/6 blastocysts, and heterozygous offspring mice were crossed to homozygosity. ifit1 2/2 mice were generated from c57bl/6 es cells lacking the whole coding region of ifit1 (20); es cells were obtained from the nih knockout mouse project (komp, allele ifit1 tm1(komp)vlcg ). the same es cell line was independently used to generate mice in another study [18] . ifnar 2/2 mice (lacking ifnar1) were a gift of murali-krishna kaja (emory university, atlanta, ga). congenic wild-type mice were obtained from taconic farms. vesicular stomatitis virus (vsv) indiana was a gift from amiya k. banerjee, lerner research institute, cleveland, ohio. for intranasal infections, between 4610 2 and 4610 6 pfu of vsv in 35 ml of endotoxin-free pbs were inhaled by isofluorane-anesthetized 8-12 week-old mice, with pbs-only as control. for intracranial infections, 10 pfu of vsv in 30 ml of endotoxin-free pbs were injected into the brains of 6-7 week-old mice, with pbsonly as control. thereafter, mice were monitored daily (twice daily after i.c. injection) for weight loss and symptoms of disease. encephalomyocarditis virus (emcv) k strain was a gift from robert h. silverman, lerner research institute, cleveland, ohio. for intraperitoneal infections, between 25 and 5610 2 pfu of emcv in 500 ml of pbs were injected into the peritoneal cavity of mice. mice were monitored daily for weight loss and symptoms of disease. mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs by cardiac perfusion with 10 ml of pbs, followed by perfusion with 10 ml of 4% paraformaldehyde/pbs for fixation. brains were placed in 4% paraformaldehyde overnight for complete fixation, submerged in 30% sucrose/ pbs overnight for cryoprotection, and frozen in o.c.t. compound (sakura finetek usa, torrance, ca, usa). 10 mm sagittal sections were cut at 220uc in a leica cm1900 cryostat, mounted on coated slides (superfrost plus, fisherbrand, fisher scientific); membranes were permeabilized by 0.2% triton x-100/pbs treatment for 15 min. for immunohistochemistry, the envision+ dab kit (dako, carpinteria, ca) was used with antimouse ifit2/p54 [38] or anti-vsv-p protein (a gift from amiya k. banerjee, lerner research institute, cleveland, ohio) as primary antibodies. for immunohistofluorescence, anti-vsv-p or anti-neun (chemicon intl./millipore, billerica, ma) or anti-gfap (sigma-aldrich, st. louis, mo) were used; labeled brain sections were stained with alexafluor-594 secondary antibody (invitrogen/molecular probes, carlsbad, ca). for detection of apoptotic cells in brain sections, the deadend fluorometric tunel system (promega) was used according to manufacturer's instructions. all objects were then mounted with vectashield (with dapi, vector labs, burlingame, ca), and examined with a leica drm fluorescence microscope. mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs after cardiac perfusion with 10 ml of pbs. brains were separated into olfactory bulbs and the remainder of the brain, snap-frozen in liquid nitrogen (as well as livers and lungs) and rna was extracted using trizol reagent (invitrogen). dnase i treatment (dnafree, applied biosystems/ ambion) and reverse transcription with random hexamers (improm-ii, promega) were performed according to manufacturer's instructions. 0.5 ng of rna was used in 384 well-format realtime pcrs in a roche lightcycler 480 ii using applied biosystem's sybr green pcr core reagents. pcr primers for murine isg49/ifit3, isg54/ifit2, isg56/ifit1 and 18s rrna have been published previously [17] ; primers targeting murine ifnb1 [59-cttctccgtcatctccataggg-39 [39] , with the alternative reverse primer: 59-cacagccctctccatcaact-39], vsv n rna [40] or emcv 3d polymerase genomic region [41] were described previously. primers for ccl2, il1b, il6, tnf, il12b and nos2 have been described previously [42, 43] . average expression levels, relative to 18s rrna and normalized by use of calibrator samples, were graphed with prism 5.02 software. for analysis of different regions of the brain, brains without ob of perfused mice were separated into cortex, cerebellum, brain stem and remaining ''midbrain'', and tissue was submerged into rnalater stabilizing reagent (qiagen) overnight and frozen. rna was then extracted via trizol and further processed and assayed by realtime rt-pcr as described above. for microarray analysis, trizol-extracted and dnase i-treated rna was additionally purified using spin columns (rneasy mini kit, qiagen) before subjection to mrna expression microarray analysis via illumina mouse ref-8 v2 beadchip and genomestudio software v2010.2 (illumina, inc.); rna hybridization to chips was performed by the lerner research institute genomics core at the cleveland clinic. microarray raw data were deposited in the ncbi gene expression omnibus (geo), accession number gse33678. for quantification of infectious vsv in organs, mice were anesthetized with pentobarbital (150 mg/kg) and blood was removed from organs by cardiac perfusion with 10 ml of pbs. organs were snap-frozen in liquid nitrogen, weighed, pestle/tubehomogenized (kimble/kontes) in 1 ml of pbs per brain or peripheral organ or 0.1 ml per pair of olfactory bulbs, and virus was titered in 10-fold serial dilutions on vero cells by plaque assay. results are expressed as plaque-forming units (pfu) per gram of tissue. for quantification of infectious vsv yields in mef, cells (2/+ifn-b pretreatment as indicated) were infected with vsv inoculum for 1 h, and after another 12 h, cells were freeze/ thawed, and cleared supernatants of lysates were assayed for vsv by plaque assay on vero cells. primary murine embryonic fibroblasts (mefs) were stimulated with 1000 u/ml murine ifn-b (pbl, inc., piscataway, nj) for 16 h and lysed in lysis buffer [50 mm tris ph 7.6, 150 mm nacl, 0.5% triton x-100, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 5 mm sodium pyrophosphate, 10 mm bglycerophosphate and 16 complete edta-free protease inhibitor (roche, indianapolis, in)]. 10 mg of whole cell extract were separated via 10% sds-page, transferred to pvdf membranes, blocked with 5% dry milk in tris-buffered saline/0.05% tween-20 overnight and labeled with anti-ifit3/p49, anti-ifit2/p54 or anti-ifit1/p56 polyclonal rabbit sera [17, 38] . single-stranded vsv leader rna (nucleotides 1-18) was t7 polymerase-transcribed in presence of [a-32 p]-ctp, yielding radiolabeled 59-triphosphorylated (ppp-) rna, followed by alkaline phosphatase treatment for generation of 59-hydroxyl (ho-) rna. ppp-rna or ho-rna were added to bacterially expressed and purified 6xhis-tagged ifit1 or ifit2 protein in reaction buffer (50 mm tris ph 8.0, 100 mm nacl, 1 mm edta, 2 mm dtt, 0.05% triton x-100, 10% glycerol) and incubated for 30 min on ice. reaction products were separated by 6% native polyacrylamide gel electrophoresis followed by exposure to film. statistical significance of mouse survival differences was calculated by mantel-cox log rank test. to assess significance of differences in gene expressions or virus titers, the two-tailed mann-whitney test was used. all calculations were performed using graphpad prism 5.02 software. previously published transcript sequences in the ncbi entrez nucleotide database: ifit2, nm_008332; ifit1, nm_008331; ifit3, nm_010501; ifnb1, nm_010510; ifnar1, nm_010508. figure s1 survival of wt and ifit2 2/2 mice after infection with low emcv dose (25 pfu). statistical significance of survival differences is indicated by p-value; n.s., not significant. (pdf) figure s2 enhanced isg and ifn-b induction in intranasally vsv-infected ifit2 2/2 brain regions. ifn-b-, and ifit3/2/1 mrna levels in different regions of brains of uninfected or vsv-infected wt and ifit2 2/2 mice at 6 d.p.i., plotted as mean+sd. n = 4 mice per infected group; nd, not done. infections were intranasal with 4610 2 pfu of vsv. (pdf) figure s3 gene induction in brains after vsv or emcv infections. a, mrna levels of select genes in brains (without obs) of uninfected or intranasally vsv-infected wt and ifit2 2/2 mice at 6 d.p.i., plotted as mean+sd; n = 3 mice per infected group; infection was intranasal with 4610 2 pfu of vsv. b, mrna levels of select genes in brains (without obs) of uninfected or intracranially vsv-infected wt and ifit2 2/2 mice at 24 h post injection, plotted as mean+sd; n = 4 mice per infected group; infection was intracranial injection with 10 pfu of vsv. c, ifit2, ifit1, ifn-b and emcv rna levels in brains 4 days after emcv infection (5610 2 pfu, n = 3 mice per infected group). (pdf) figure s4 region-selective induction of apoptosis in brains of intranasally vsv-infected ifit2 2/2 mice. ifit2 2/2 mice were i.n. infected with 4610 2 pfu of vsv; at 6 d.p.i., adjacent sections of fixed brains were labeled to detect apoptotic cells (tunel) or vsv p protein (immunohistofluorescence), n = 2 mice; only few regions such as striatum show positive tunel; infected wt brains and uninfected control brains of either genotype did not show appreciable signals, hence data not shown). (pdf) figure s5 vsv yields from infected wt and ifit2 2/2 mef. immortalized mef were treated for 16 h with 10 u/ml ifn-b and infected with vsv at moi 10. 12 hours after infection, virus yields were determined by plaque assay. results are plotted as mean+sd on log scale, representing one of two independent experiments. (pdf) figure s6 murine ifit2 protein does not bind ppp-rna. single-stranded radiolabeled vsv leader rnas (nt 1-18) with either 59-triphosphorylated or free 59-hydroxyl-ends (ppp-rna or ho-rna) were in vitro incubated with purified murine ifit1 ( = p56) or ifit2 ( = p54) proteins; formation of protein/rna complex was detected by electrophoretic mobility shift assay. (pdf) table s1 enhanced gene expression in brains incl. obs of intranasally vsv-infected ifit2 2/2 versus wt mice at 6 d.p.i. wt or ifit2 2/2 mice were intranasally vsv-infected with 4610 2 pfu, and at 2 or 6 d.p.i., brain (incl. ob) rna expression profiles were obtained by microarray. genes are ranked by their ''fold expression level in ifit2 2/2 over wt at 6 d.p.i.''. only genes with at least 3-fold higher expression level in ifit2 2/2 are included. note: the ifit1/isg56 probe of the illumina mouse ref-8 chip is defective and therefore the gene is not included in this list. (pdf) local type i ifn receptor signaling protects against virus spread within the central nervous system functional role of type i and type ii interferons in antiviral defense a null mutation in the gene encoding a type i interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses differential roles of mda5 and rig-i helicases in the recognition of rna viruses distinct and essential roles of transcription factors irf-3 and irf-7 in response to viruses for ifn-alpha/beta gene induction essential role of ips-1 in innate immune responses against rna viruses modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything dcs and nk cells: critical effectors in the immune response to hiv-1 pathogenesis of murine coronavirus in the central nervous system targeted disruption of the mouse stat1 gene results in compromised innate immunity to viral disease targeted disruption of the stat1 gene in mice reveals unexpected physiologic specificity in the jak-stat signaling pathway identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays interferon-inducible antiviral effectors a diverse range of gene products are effectors of the type i interferon antiviral response the isg56/ifit1 gene family induction and mode of action of the viral stressinducible murine proteins, p56 and p54 novel characteristics of the function and induction of murine p56 family proteins ifit1 is an antiviral protein that recognizes 59-triphosphate rna coordinated regulation and widespread cellular expression of interferonstimulated genes (isg) isg-49, isg-54, and isg-56 in the central nervous system after infection with distinct viruses -o methylation of the viral mrna cap evades host restriction by ifit family members convenient assay for interferons vesicular stomatitis the earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system distribution of vesicular stomatitis virus proteins in the brains of balb/c mice following intranasal inoculation: an immunohistochemical analysis interferons alpha and beta as immune regulators-a new look human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses the inhibitory action of p56 on select functions of e1 mediates interferon's effect on human papillomavirus dna replication interferon-inducible protein, p56, inhibits hpv dna replication by binding to the viral protein e1 alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication inhibition of vesicular stomatitis viral mrna synthesis by interferons host immune response to vesicular stomatitis virus infection of the central nervous system in c57bl/6 mice vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity autophagydependent viral recognition by plasmacytoid dendritic cells subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus tissue-specific and inducer-specific differential induction of isg56 and isg54 in mice cell-specific irf-3 responses protect against west nile virus infection by interferondependent and -independent mechanisms replication and propagation of attenuated vesicular stomatitis virus vectors in vivo: vector spread correlates with induction of immune responses and persistence of genomic rna rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction interleukin-12 (il-12), but not il-23, deficiency ameliorates viral encephalitis without affecting viral control factors supporting intrathecal humoral responses following viral encephalomyelitis we thank michifumi yamashita and niranjan butchi for sharing technical expertise. key: cord-003667-u1xa44nw authors: rodriguez, sergio e.; cross, robert w.; fenton, karla a.; bente, dennis a.; mire, chad e.; geisbert, thomas w. title: vesicular stomatitis virus-based vaccine protects mice against crimean-congo hemorrhagic fever date: 2019-05-23 journal: sci rep doi: 10.1038/s41598-019-44210-6 sha: doc_id: 3667 cord_uid: u1xa44nw crimean-congo hemorrhagic fever virus (cchfv), a tick-borne bunyavirus, can cause a life-threatening hemorrhagic syndrome in humans but not in its animal host. the virus is widely distributed throughout southeastern europe, the middle east, africa, and asia. disease management has proven difficult and there are no broadly licensed vaccines or therapeutics. recombinant vesicular stomatitis viruses (rvsv) expressing foreign glycoproteins (gp) have shown promise as experimental vaccines for several viral hemorrhagic fevers. here, we developed and assessed a replication competent rvsv vector expressing the cchfv glycoprotein precursor (gpc), which encodes cchfv structural glycoproteins. this construct drives strong expression of cchfv-gp, in vitro. using these vectors, we vaccinated stat-1 knock-out mice, an animal model for cchfv. the vector was tolerated and 100% efficacious against challenge from a clinical strain of cchfv. anti-cchfv-gp igg and neutralizing antibody titers were observed in surviving animals. this study demonstrates that a rvsv expressing only the cchfv-gp has the potential to serve as a replication competent vaccine platform against cchf infections. crimean-congo hemorrhagic fever virus (cchfv) is a highly pathogenic zoonotic agent in the orthonairovirus genus within nairoviridae 1 . the principal reservoirs for cchfv are ixodid hard ticks primarily belonging to the genus hyalomma 2 . these ticks maintain the virus in nature by feeding on small mammals, ungulates, and birds within thirty countries across the eastern hemisphere 3 . human infection can occur from the bite of an infected tick, exposure to infected animal products, or through nosocomial transmission 4 . cchfv case-fatality rates in most outbreaks range from 3-30%, though higher rates have been documented in some instances 3, 5 . cchfv is categorized as a category a priority pathogen by the us national institutes of health due to its associated morbidity and mortality, potential for public health/societal impact, as well as a lack of approved therapeutic options or us/eu licensed vaccines for treatment. the guanosine analogue ribavirin has been suggested as a therapeutic when given early in human infections; however, efficacy has not been clearly demonstrated in clinical trials for cchf 6 . for these reasons, vaccines and therapeutic countermeasures against cchfv are currently under development. full or partial mature cchfv particles contain single stranded, tri-partite, negative sense rna genomes with small (s), medium (m), and large (l) segments, respectively encoding the structural nucleoprotein (np), two envelope proteins (g n and g c ) and the viral rna-dependent-rna-polymerase (rdrp) 3 . the m-segment contains a 5.1 kilobase open-reading frame which codes for a glycoprotein precursor polypeptide (gpc) 7 . host cell processing, cleavage events, and post-translational modifications of this gpc yield the two mature structural glycoproteins g n and g c , along with several non-structural glycoproteins which aid in structural g n and g c maturation [7] [8] [9] [10] [11] [12] . the two glycoproteins are likely responsible for pertinent events in the viral replication cycle such as viral attachment, cell entry, tissue tropism(s), and induction of protective immune response as seen similarly with other members of bunyavirales 13, 14 . the latter has been demonstrated for cchfv using monoclonal antibodies (mab) directed against g n and g c , which have demonstrated in vitro neutralization in tissue culture and in vivo passive protection in suckling mice [13] [14] [15] . these data suggest that the gpc would be an important antiviral target for therapeutic and vaccine efforts. our initial attempts using the dna clone recovery system designed by lawson et al., failed to produce infectious δgrvsv with cchfv-gpc (fig. 1a) 39 . to overcome this, we used a modification based on a system described by whitt 40 relying on in trans vsv glycoprotein (g) complementation (vsv-g*) of δgrvsv virions. this technique allowed for vsv-g, incorporation into recoveries to facilitate efficient assembly of the rvsvδg-cchfv-gpc genome without the need for cchfv-gpc to participate in initial infection of recovered virions (fig. 1a) . we recovered a virion containing the cchfv-gpc in the genome with vsv-g complementation (designated vsv-g*-δgrvsv-cchfv-gpc), which contributed to a single-cycle infection; unless vsv-g is provided in trans this virus will not replicate effectively in cell culture. after the initial recovery of vsv-g*-δgrvsv-cchfv-gpc, this virus was passaged on vsv-g complemented bhk cells and passaged onto un-complemented ('normal') bhk cells. we were unable to isolate infectious virus from initial supernatants, however, seven total serial passages of supernatants on un-complemented bhk cells resulted in eventual cytopathic effect (cpe) in cell culture with foci/plaque formations appearing on the monolayers. these monolayers with cpe were harvested for rna and were stained for cchfv-gpc antigens via immunofluorescence assay and found to be positive (data not shown). this replication competent construct was designated δgrvsv-cchfv-gpcδ (fig. 1b) . sanger sequencing of both constructs, using primers for the vsv backbone and cchfv-gpc orf, was carried out which confirmed a rvsvδg-cchfv-gpc genome and revealed several single nucleotide polymorphisms (snp) (data not shown). next generation sequencing (ngs) was then performed to confirm sanger results and further detail the snps within the entire genomes of both constructs (supplemental table 1 ). ngs sequencing demonstrated fourteen identical single nucleotide polymorphisms (snp) preserved between the replication deficient and replication competent constructs; with the replication competent construct possessing four additional non-synonymous mutations within the cchfv-gpc (fig. 1b , supplemental table 1 ); henceforth referred to as gpcδ. two of these mutations within the cchfv-gpc of the replication competent construct, resulted in the truncation of fourteen amino acids off the c-terminal tail of the g c (fig. 1b) . to assess the growth kinetics compared to authentic cchfv, we performed single-cycle growth curve analysis on bhk cells infected with respective viruses at various time intervals up to 96 hrs post infection (hpi) or full monolayer destruction. the rvsv-gfp (wild-type control) peaked in titer at approximately 24 hpi, while cchfv prototype-strain: ibar10200 peaked at 48 hpi ( fig. 2a) . the replication competent pseudotype, δgrvsv-cchfv-gpcδ had peak titer, at approximately 36 hpi ( fig. 2a) . to assess the expression of the cchfv-gpc in the vector, we performed an immunofluorescence assay, using mabs that bind to either the cchfv-g c or -g n . the assay revealed strong in vitro expression of both antigens (fig. 2b) . we additionally examined if cchfv-g c was incorporated onto the rvsv virion. we used coomassie staining and western blot analysis on gradient purified δgrvsv-cchfv-gpcδ and semi-purified rvsv-gfp and cchfv (as controls). through coomassie staining, differences in number and mobility of protein bands were detected among the wt rvsv-gfp and δgrvsv-cchfv-gpcδ (fig. 2c , lanes 1, 2). the cchfv-gpc recombinant had additional protein bands below 20 kda with analogous bands detected in cchfv virion pellets (fig. 2c , lanes 2, 3). the cchfv-gpc recombinant also possessed more pronounced protein bands at 60 kda, compared to our wt rvsv-gfp (fig. 2c , lanes 1, 2). cchfv structural glycoproteins g n and g c present as bands of approximately at 37 kda and 75 kda, respectively (fig. 2c , lane 3), on sds-page gels 7 . there was no observable g n band found on the cchfv-gpc recombinant at 37 kda; however, there were two distinct bands between 60-80 kda in the recombinant (fig. 2c, lanes 2) . the δgrvsv-cchfv-gpcδ did not encode for a vsv-g (which was confirmed by our deep sequencing shown in supplemental table 1 ), nor was it complemented with vsv-g after seven rounds of bhk passaging. a duplicate protein gel was run and further probed by western blotting using a mab previously identified to be specific for cchfv-g c by western blot (mab 11e7, bei resources) 14 as there is currently, no available antibody that probes solely mature g n by western blot. these western blot data demonstrated limited, non-specific binding to three vsv proteins as observed in lanes rvsv-gfp (fig. 2d , lane 1), but showed strong signal to at least two antigens at approximately 75 kda and 150 kda for δgrvsv-cchfv-gpcδ (fig. 2d, lane 2 ). this indicated that mature cchfv-g c and potentially a precursor molecule or an oligomeric form of cchfv-g c (due to the lack of β-mercaptoethanol in antigen preparations), are incorporated in/on δgrvsv-cchfv-gpcδ virions. the semi-purified cchfv also showed limited non-specific binding to antigens at 57 kda (cchfv-np) and 37 kda (cchfv-g n ), but distinct signal at 75 kda (cchfv-g c ) (fig. 2d , lane 3). to examine the ultrastructure of the δgrvsv-cchfv-gpcδ, transmission electron microscopy studies were conducted. particles of rvsv-gfp ('wild-type' vsv electron microscopy control) were observed between 170-200 nm (fig. 2e , top). consistent with other rvsv pseudotyped with bunyavirus gp 28 , our δgrvsv-cchfv-gpcδ pseudotype maintained rhabdovirus morphology and classical bullet shape with coiled intra-virion structure (fig. 2e , bottom). particles were observed to have lengths ranging between 210-260 nm (fig. 2e , bottom). this apparent length increase of the δgrvsv-cchfv-gpcδ is likely due to the genome containing approximately 4,000 extra nucleotides in comparison to the rvsv-gfp genome (which is also slightly larger than native wild-type vsv indiana [genbank number nc_001560.1]). additionally, immunolabeling of δgrvsv-cchfv-gpcδ was employed with mab to cchfv-g c and counterstained with 15 nm gold conjugated secondary mab (fig. 2e, bottom) . immunolabeling demonstrated labeling of g c spikes on the virion surface of δgrvsv-cchfv-gpcδ (fig. 2e, bottom) . with the data supporting that the replication competent construct expressed cchfv-gp in vitro and additionally expressed cchfv-g c on the surface of the virion, an in vivo study was designed to test the ability of the construct to function as an experimental vaccine. the stat-1 −/− mouse model for cchfv was selected to test these constructs for protective efficacy 19 . in pilot studies (supplemental fig. 1 ) the replication deficient construct (vsv-g*-δgrvsv-cchfv-gpc), failed to provide protection; however the replication competent (δgrvsv-cchfv-gpcδ) construct did demonstrate some protection depending on dose. based on the results of these initial pilot studies, we elected to adjust vaccine and challenge doses, and administered 10 7 pfu/dose of the replication competent virus (δgrvsv-cchfv-gpcδ) to prime and boosted groups of five stat-1 −/− mice, respectively (fig. 3) . as a control, we used pbs as a mock vaccination. a single mouse succumbed in the boosted group (leaving only four mice to be challenged in the boosted group) the day of boosting. at 35 days post prime, all mice were challenged with 50 pfu of cchfv strain turkey2004 and were monitored for clinical signs, weights, and temperatures. we challenged with a human/clinical strain, turkey200406546, designated throughout this work as turkey2004; which has previously been published in genbank with the accession numbers ky362517 (s-segment), ky362519 (m-segment), and ky362515 (l-segment) 41 . mean time-to-death (mtd) was 5.6 days post infection (dpi), with a standard deviation (sd) +/− 0.55 dpi as demonstrated with the pbs control group (fig. 4a ). for both prime and boosted groups, 100% protection was observed out to 35 dpi (fig. 4a) . the prime www.nature.com/scientificreports www.nature.com/scientificreports/ and pbs group displayed weight loss, while the boosted group did not show weight loss (fig. 4b ). all three groups displayed elevated temperatures for the first three days post challenge (fig. 4c) . these results indicate that the primed group showed a marked illness, while the boosted group did not display observable disease. to examine the humoral response in vaccinated mice, sera from these groups were analyzed for both igg to cchfv-gp and for the presence of cchfv neutralizing antibodies. a separate group of animals was vaccinated (n = 5, per group) at similar prime/boost doses and bled at days −35 and −21 to assess pre-challenged immune status. circulating iggs to cchfv-gp were detected in sera of both vaccinated groups, beginning at day −21 and were found to have increased substantially at the end of study (fig. 5a) . curiously, at the study end point, the prime group displayed higher titers compared to the prime plus boosted group (fig. 5a ). this indicates the prime only group had a higher concentration of igg at the study endpoint which recognized cchfv-gp, compared to the boosted group. the reciprocal titer of the pbs group had negligible igg responses to cchfv-gp (fig. 5a) . to determine the neutralizing activity of these sera, a plaque reduction neutralization test (prnt) was carried out using the turkey2004 challenge isolate. as depicted in fig. 5b , the prime only group had neutralizing antibody titers with a prnt 50 of < 1:1,280 and the boosted group a prnt 50 of < 1:320 at the study endpoint (fig. 5b ). our prnt positive control, hyper-immune mouse ascitic fluid (hmaf, kindly provided by t. ksiazek, galveston, texas) raised against cchfv exposed mice, showed a prnt 50 of < 1:160 while the pbs control mice did not demonstrate a prnt 50 value (fig. 5b ). particle preparations were stained with α-cchfv-g c mab 11e7 using an hrp-conjugated secondary. (e) transmission electron micrographs of rvsv-gfp and replication competent δgrvsv-cchfv-gpcδ particles from bhk cells, semi-purified using a 20% sucrose cushion or purified using iodixanol gradient centrifugation, negatively stained with 2% aqueous uranyl acetate, immunolabeled with α-cchfv-g c mab 11e7 and a secondary 15 nm gold labeled secondary. samples were fixed with 2% glutaraldehyde. images were adjusted for brightness, contrast, and formatted for size for display purposes. coomassie gel and western blot images were cropped from the same image file at same imaging parameters for ease of viewing. gel and blots originals are available in supplemental information. other original raw data files available upon request. www.nature.com/scientificreports www.nature.com/scientificreports/ additionally, at study endpoints for each cohort, we examined tissues by immunohistochemistry (ihc) for the cchfv-np antigen. the control cohort had marked cchfv-np immunolabeling in hepatocytes within the liver sections (fig. 6a ) while liver sections from the prime only and boosted cohorts had no observable www.nature.com/scientificreports www.nature.com/scientificreports/ cchfv-np immunolabeling (fig. 6a,b) . we further examined the spleen tissue sections and observed that the control cohort had marked cchfv immunolabeling in mononuclear cells (fig. 6d) , and that the prime cohort had a cytoplasmic, mild, and diffuse immunolabeling of mononuclear cells primarily in the red pulp (fig. 6e ). the boosted cohort had no specific cchfv immunolabeling within the spleen sections (fig. 6f ). studies have had varied success expressing cchfv-gpc in trans on viral vectors 42, 43 . rvsv with cchfv-gpc has likely been challenging due to the numerous post-translational modifications required to mature, and yield functional, cchfv-gpc 7,8,10,12 . additionally, the cchfv-gpc can form immature cchfv particles at the golgi and egress via vesicular transport 14, 44 . unlike cchfv, vsv buds from the plasma membrane 45 , which hampers efforts to recover a replicating δgrvsv vector with cchfv-gpc expressed. we used a mammalian www.nature.com/scientificreports www.nature.com/scientificreports/ codon optimized cchfv-gpc which has demonstrated expression of cchfv-g c to the cell surface 20 , while maintaining native cchfv-gpc maturation factors. we hypothesized that this expression due to codon optimization could facilitate shuttling of cchfv-gp to the plasma membrane where vsv could acquire and bud with functional components of the gpc in the viral envelope. although we were able to drive strong expression cchfv-gpc in vitro, we were unable to generate a replication competent pseudotype virus without initially using vsv-g* in trans. there have been multiple studies that have examined pseudotyping rvsv with a cchfv-gpc using a plasmid encoding a vsvδg backbone. a luciferase reporter pseudotype with in trans expression of the cchfv-gpc was developed in 293 t cells by shtanko et al. 43 . suda et al., also performed similar pseudotyping with in trans expression in 293 t cells with various full length gpc and modified gpc constructs containing truncated g c c-terminal/endodomain tails 42 . these pseudotypes also had a luciferase or gfp orfs genomically encoded in the vsvδg backbone 42 . both published pseudotyped cchfv-gpc systems were used for neutralization or entry/infection studies; however, these constructs were not characterized by western blot or immune electron microscopy to understand what is on the pseudotype envelope; nor were either constructs self-replicating or capable of further expressing antigen elements of cchfv-gpc post infection 42, 43 . with the tools currently available, we were able to detect by western blot and electron microscopy, a form of cchfv-g c that was present and functional on the surface of the δgrvsv-cchfv-gpcδ virion. the current situation with reagents to mature cchfv-g n limit assaying for this particular antigen, as commercially available antibodies only target the precursor molecule cchfv-preg n . immune electron microscopy demonstrated that cchfv-g c was incorporated on the surface of the virion. while we were able to immunolabel for cchfv-g c , page and coomassie staining analysis of our purified virion lysates did not reveal prominent protein bands at the 37 kda position, the estimated size of mature g n 7 . nonetheless, we did have three smaller protein bands in the δgrvsv-cchfv-gpcδ preparations. similar protein profiles have been shown in other vectored cchfv-gpc preparations, as shown by buttigieg et al., 2014 . it is possible that these protein bands may also correspond to vsv-m2 and -m3 by alternative initiation at downstream start codons present in the orf of vsv-m 46 . while not the focus of this current study, these observations warrant further characterization should this vector be used as a tool to further study cchfv-gpc mechanisms. suda et al., have shown an increase in the amount of infectious pseudotype the more the cchfv-g c tail is truncated, up to a deletion of 53 residues at the end of the c-terminal cchfv-g c tail 42 . our data supports this region as a probable, or at the very least, contributory mechanism which enables the replication competent δgrvsv-cchfv-gpcδ pseudotype formation, as our pseudotype also was found to contain a c-terminal cchfv-g c tail truncation (fig. 1b) . there is likely a localization or retention signal within the transmembrane region or endodomain tail which is aberrated and fails to transport cchfv-g c solely to the intracellular compartments; this may in turn permit shuttling to the plasma membrane. once again this is not the focus of this study, although it will be examined in the future. it is interesting to note, that a similar motif has been demonstrated by other bunyaviruses 47, 48 and has also been demonstrated with other vsv pseudotypes 49 . structural cchfv-g c has been shown to be an important protein for cchfv and contains a putative receptor binding region for mammalian cell surface nucleolin 50 , a class ii viral fusion domain 51 , a neutralization epitope 13 , and human linear b-cell epitope sites 52, 53 . our replication competent rvsv vector can enable further exploration of these components at lower containment (bsl-2), without the need for transfections for in trans expression of cchfv-gpc onto vsvδg systems. when exploring the use of rvsv expressing cchfv-gp as potential vaccines in immunocompromised mice, there was a concern of murine virulence, which has been observed for wild-type vsv 54, 55 . studies with rvsv expressing hemorrhagic fever virus gp have demonstrated lethal outcomes in the stat-1 −/− model 56 . this has hampered the stat-1 −/− animal platform from serving as a vaccine development tool, as 'vaccinated' mice succumb to a prime dosing 56 . because of this information we conducted several pilot studies examining effective dosing and challenge strategies, for both the replication deficient (vsv-g*-δgrvsv-cchfv-gpc) and replication competent (δgrvsv-cchfv-gpcδ) construct. demonstrating the safety profile of both constructs (supplemental fig. 1) , neither group had attributed fatality due to the vaccination; however, only our replication competent construct demonstrated protection. in selecting the cchfv challenge isolate, we chose an isolate with low passage history and demonstrated clinical relevance (i.e., documented history of disease in humans). the turkey2004 isolate, came from a clinical case 41 . elisa and prnt experiments on study endpoint sera, demonstrate a humoral igg response to cchfv-gpc with observed neutralizing antibodies produced from the prime group that received a high (10 7 pfu) dose of δgrvsv-cchfv-gpcδ (fig. 5a,b) . curiously, our boosted (10 7 pfu) group had lower detectable antibodies to the cchfv-gpc with lower neutralization titers (fig. 5a,b) . clinical data for these groups following cchfv challenge show that the prime only group had elevated temperatures and weight loss, additionally two animals from this cohort displayed clinical signs including roughed fur indicating illness from the cchfv challenge (fig. 4b,c, data not shown) . while the boosted group did display elevated temperatures, there was neither substantial weight loss detected nor observable changes in clinical scoring for any of the animals (fig. 5b ,c, and data not shown). further analysis of tissues at study end point for these cohorts also revealed that while there was no immunolabeling in the liver of the vaccinated animals, the prime cohort had diffuse cytoplasmic immunolabeling of mononuclear cells in the spleen sections (fig. 6e ). this finding, along with the clinical scores, suggested that there was more replication of cchfv in the prime group compared to the boosted group. this could potentially lead to the higher anti-cchfv-gpc igg titers in the prime cohort at the study endpoint as the cchfv challenge may have served as a heterologous booster, increasing levels when compared to the δgrvsv-cchfv-gpcδ boosted cohort (fig. 5a,b) . regardless, protection was achieved by both regimens, although the boosted group data suggests that at study endpoint, the observed igg titers against cchfv-gpc along with lower neutralizing titers (prnt 50 of < 1:320) are evidence of the ability to combat lethal cchfv infection in the stat-1 −/− mouse model after vaccination (fig. 5a,b) . www.nature.com/scientificreports www.nature.com/scientificreports/ correlates of protection against cchf have been difficult to define due to the multiple vaccine and delivery platforms examined to date 16 . several cchfv experimental vaccines studies have identified cell-mediated and humoral involvement, with some instances of neutralizing antibody production 24, 57 . in looking at what is known for human cchf, survivors mount a humoral response whereas those who succumb, typically lack an igg response 3 . other studies have examined antibody and neutralizing responses from the various vaccine platforms. dna vaccines following a three round vaccination regimen have induced detectable antibodies with neutralizing capacity observed up to 1:160 in prnt dilutions and achieved 100% protection, depending on the antigens encoded on the dna plasmids 25, 58 . a cell culture based inactivated virus vaccine achieved high igg titers (1:102,400) and a high neutralizing response of 1:1,024; however, this also required three vaccination rounds, an alum adjuvant, and conferred 80% protection against the clinical isolate turkey-kelkit06 in ifnar mice 21 . antibody titers, neutralization capacity, and challenge virus presented here were similar, though through the vsv platform, we achieved greater protection with a single dose. these studies, along with our own, suggest other facets of the immune system are likely involved in conferring complete protection. as this study was a proof of concept study and not a correlate study, t cell responses were not evaluated, and could be contributory as other groups have shown 23, 25, 26 . now that we have established that this new vector can provide protective benefit, our future studies will temporally examine the antibody and t cell repertoire after prime and boosting doses following δgrvsv-cchfv-gpcδ, but before cchfv challenge, as these would be informative for the stat-1 −/− mouse model. in the future, the use of the is murine model might provide additional insights using an intact murine immune systems for vaccinations with a lethal model for cchfv infections 20 . ideally, an immunocompetent, larger animal model is greatly needed in the cchf field to further test the array of cchfv experimental vaccines which have shown promise in these mouse models. a promising step forward appears to be the nonhuman primate disease model that has been recently developed 59 . however, cchf in this model appears strain and challenge route dependent. in conclusion, this study offers not only a tool to study the biology off cchfv as it relates to structural g c , but also serves to develop and characterize a replication competent pseudotype, in relation to cchf vaccine development. the replication competent construct provides up to 100% protection with an observed humoral response, with a single-injection from a human isolate challenge strain. this information is valuable in designing future studies in cchfv animal models, and establishes characterized tools to examine the biology of structural cchfv-g c in a pseudotyped rvsv system. ksiazek, utmb -world reference center for emerging viruses and arboviruses, galveston, tx), were propagated in vero e6 cells once, plus previous passages in suckling mice and vero cells since isolation. all in vitro and in vivo work with cchfv was performed in a biosafety level 4 facility at the galveston national laboratory, university of texas medical branch, galveston, tx. all cell and viral stocks were tested and free of mycoplasma, by pcr kit (intronbio, gyungg-do, south korea). monoclonal antibodies (mab) mouse-α-cchfv-g c 11e7 and 8a1, and mouse-α-cchfv-preg n antibody 13g8 were generated and characterized as described previously 13, 14 . described antibodies are available at bei resources (atcc) except for 8a1, which was kindly provided by united states armed forces research institute for infectious diseases, frederick, md. generation of δgrvsv vectors. the rvsv vector was cloned and recovered from cdna as previously described 60 . briefly, a bluescript backbone plasmid under t7 polymerase promoter control encoding a δg, vsv indiana backbone expressing a chimeric zaire ebolavirus (chebov) glycoprotein (gp), was used as the vector (designated pvsvδg-chebov-gp-3). this plasmid was modified by cutting out an existing chebov-gp coding sequence via mlui and nhei restriction sites, yielding a pvsvδg vector. an insert, coding for the codon optimized open reading frame of the complete cchfv glycoprotein precursor (gpc), was digested and ligated into the pvsvδg vector. the cchfv-gpc insert was created by overhang pcr mutagenesis; flanking a 3′ mlui restriction site plus kozak sequence, and a 5′ xbai restriction site, from a pcr amplified, codon optimized, pcagg-cchfv-gpc (kindly provided by j. kortekaas, central veterinary institute, lelystad, netherlands). this ligated and cloned plasmid, designated pvsvδg-cchfv-gpc, was transfected into bhk cells that were also co-transfected with vsv protein n, p, g, and, l 'helper' plasmids under t7 promoter control and driven by infection (moi 5) with recombinant vaccinia virus expressing t7 polymerase (rvv-tf7-3; kindly provided by m. whitt). recovered virus, designated vsv-g*-δgrvsv-cchfv-gpc, was collected 24-48 hrs post infection/ transfection, filtered twice through a 0.2 μm filter to remove vaccinia virus, plaque purified onto vsv-g* complemented bhk cells, and stored at −80 °c for further use. all plasmid maps and cloning primer sequences are available upon request. infections, enumeration, growth kinetics, and preparation of viral material. for infections of replication deficient vector, semi-confluent (~60-80%) monolayers of bhk cells were transfected (lipofectamine www.nature.com/scientificreports www.nature.com/scientificreports/ ltx, invitrogen) with 1 μg/1 × 10 6 cells using pcagg-vsv-g (indiana) . once monolayers displayed cell rounding and syncytia, they were infected at an moi of 0.1 with vsv-g*-δgrvsv-cchfv-gpc. supernatants were harvested at 24 hrs post infection (hpi) and clarified at 2,000 rpm for 10 min at 4 °c. confluent monolayers of bhk cells were infected with replication competent δgrvsv-cchfv-gpcδ at moi 0.1 for 1 hr at 37 °c with 5% co 2 with rocking at 15 min intervals and harvested/clarified at 48 hpi. plaque assays for viral titrations were carried out in an analogous manner, with an overlay media final concentration of 1.25% avicel rc-581 (fmc biopolymer, philadelphia, pa) in 1x eagles minimum essentials medium (mem) with 5% fbs and 1% p/s on bhk cells for 24-48 hpi. after incubations, overlays were aspirated and a 10% buffered formalin fix with a 1x crystal violet stain was incubated onto monolayers for one hr. plaques were enumerated and plaque forming units (pfu) were determined by averaging technical replicates per sample. single-cycle growth curves at moi 0.1 were performed by absorbing rvsv-gfp, δgrvsv-cchfv-gpcδ, or cchfv onto duplicate monolayers of bhk cells in six-well plates as described above. inoculum was aspirated, cells were washed three times with pbs, and d5 was added to monolayers and incubated at various time points indicated in fig. 2a . at designated time points, samples were collected, clarified, and stored at −80 °c until titrations were performed described above. cchfv-g c protein analysis. immunofluorescence analysis was carried out by infecting huh-7 monolayers at a moi of 0.1 for 24 hpi. cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 1% triton x-100 and stained with 1:500 diluted mab 8a1 or 13g8. cells were washed, blocked with 5% bsa, and incubated with a dilution of 1:1,000 secondary goat-α-mouse mab conjugated to fluorescein isothiocyanate (fitc) (invitrogen). cells were mounted and counterstained with the nuclear stain of 4′,6-diamidino-2-phenylindole (dapi) (invitrogen). stained cells were examined on a nikon eclipse ti-s fluorescence microscope. whole virions were analyzed by infecting bhk cell monolayers, semi-purifying clarified supernatants over a 20% sucrose cushion at 37,000 rpm for 45 min at 4 °c using a beckman sw 41 ti rotor. viral pellets were lysed using np-40 with 1x protease inhibitor cocktail (invitrogen). recombinant vsv lysates were incubated at 56 °c for 10 min and protein subsequently quantified using bca protein assay per manufacturers' instructions (thermo fisher scientific, waltham, ma). per institutional inactivation protocols, cchfv lysates had a modified inactivation protocol using instead 2x laemmli sample buffer (lsb) (thermo fisher scientific) at 95 °c for 15 min boiling and transfer to a fresh tube. approximately 200 ng of purified and semi-purified virion associated total protein was mixed 1:1 with 1x lsb (without β-mercaptoethanol) and run on 4-20% gradient mini-protean tgx electrophoresis gels (bio-rad, hercules, california). coomasie staining was accomplished via incubating tgx gels in coomassie fluor orange protein gel stain (thermo fisher scientific) per manufacturers' instructions and imaged at 300 nm on a gel doc xr+ gel documentation system (biorad). western blots were run on tgx gels using wet tank transfer to hybond-p polyvinylidene difluoride (pvdf) membranes (ge healthcare, little chalfont, uk). membranes were blocked with 5% bsa overnight at 4 °c in tris/0.1% tween 20 (sigma-aldrich) followed by incubation with primary mab 11e7 diluted at 1:1,000, overnight at 4 °c. secondary horse radish peroxidase (hrp) conjugated goat-α-mouse antibody (thermo fisher scientific) was diluted 1:10,000 and incubated on the membrane for 1 h at room temperature. detection of hrp was accomplished via pierce ecl western blotting substrate (thermo fisher scientific), hyperfilm ecl (ge healthcare), and kodak carestream film with x-omat 2000 processor (eastman kodak company, rochester, ny). were placed in trizol ls (thermo fisher scientific) at a ratio of 1:5, mixed, incubated for 10 min at room temperature, and transferred to fresh tubes. rna was isolated from sample mixtures using zymo research direct-zol rna min-prep (zymo research corp, irvine, ca), per manufacturers' instructions. rna was quantified using a nanodrop 8000 (thermo fisher scientific) and approximately 500 ng total rna was used to create cdna using the superscript iii first-strand synthesis system (invitrogen) and a vsv-m, matrix protein gene 3′ forward primer. sanger sequencing on the cdna was performed using vsv-m, vsv-l, and cchfv-gpc (codon optimized) open reading frame primer sets and accomplished by the utmb molecular genomics core using an abi prism 3130xl dna sequencer (applied biosystems, foster city, ca). sequence analysis was performed using geneious r9 (biomattes, auckland, new zealand) based on consensus and plasmid maps. all cdna/sequencing primers and consensus/plasmid maps are available upon request. to analyze the stocks of cchfv or rvsv vaccine vectors used in this study we performed deep sequencing analysis of rna isolated from these virus stocks. briefly, viral rna was isolated from a trizol ls (invitrogen)/sample mixture using a direct-zol rna mini-prep (zymo research), per manufacturer's instructions. approximately 150 ng of purified rna was used to make cdna using the ovation rna-seq. 2.0 kit (nugen) and this in turn was used for the preparation of the double-stranded dna library, using encore ion torrent library prep kit. sequencing was performed by the utmb molecular core on the ion torrent using 318-v2 deep sequencing chips. sequence analysis was performed using dna star seqman ngen software (dna star) based on unpaired analysis of 125 base pair overlaps. ultrastructural analysis. viruses were propagated in multiple t-150 flasks of confluent bhk cells. viral supernatants were harvested and clarified as described above. clarified supernatants were concentrated by mixing with buffered 4x polyethylene glycol with incubation for 4 hrs at 4 °c, followed by centrifugation at 6,800xg for 30 mins at 4 °c. concentrated pellets were re-suspended in pbs with protease inhibitor and overlaid atop optiprep (sigma-aldrich) continuous gradients of 6-48% buffered iodixanol. viruses were banded by ultracentrifugation at 25 www.nature.com/scientificreports www.nature.com/scientificreports/ [ems], hatfield, pa) for 10-30 mins, incubated with mab 11e7 at 1:10 dilutions. antibodies were absorbed for 30 mins in wet chamber and washed with pbs containing 1% bsa, and incubated with the secondary antibody, goat-α-mouse conjugated to 15 nm colloidal gold particles (ems), at a dilution of 1:20 for 30 mins. grids were washed with pbs and 1% bsa, fixed using 2% (w/v) aqueous glutaraldehyde for 10 mins, and finally stained with 2% (w/v) aqueous uranyl acetate. grids were examined at 60 kv using a philips cm-100 transmission electron microscope. ethics of care, vaccination, and animal challenge. animal studies were approved by the utmb institutional animal care and use committee (iacuc). animal research was carried out in compliance with the animal welfare act and other federally regulated stipulations regarding animals and adherences to the guide for the care and use of laboratory animals, national research council, 2013. the animal facilities where this research was conducted are accredited by the association for assessment and accreditation of laboratory animal care international. this study used female 4 to 8 weeks old 129s6/svev-stat1 tm1rds mice (stat-1 −/− ) (taconic, germantown, ny). after an acclimatization period in barrier conditions in environmentally enriched sterile housing, mice were anesthetized by isoflurane and implanted with subdermal transponders, which provide coded identifiers and permitted body temperature measurements (biomedic data solutions, seaford, de). vaccine preparations were diluted in hanks balanced salts medium with 2% fbs, along with pbs for control groups. after anesthesia by isoflurane, 500 ul of each preparation was administered intraperitoneally (i.p.), with five stat-1 −/− mice per experimental group. clinical scoring, body temperature, and weight, were recorded daily. at 35 days post vaccination (prime), mice were challenged with 500 ul i.p. with either 100 pfu of cchfv-ibar10200 or cchfv-turkey200406546 (referred to as turkey2004) (however, 50 pfu calculated back titer of challenge preparation for the turkey2004 administration and is thus reported as such in fig. 3 ), respectively. all challenge doses were frozen for storage and verified by back-titrations by plaque assay on sw-13-cdc cells as outlined above. after challenge, animals were observed for clinical scoring, temperature, and weight change. upon reaching institutionally approved endpoint score criteria, or study end-point, blood samples were collected into k3edta containing collection tubes (granier, usa) and plasma was separated then frozen at −80 °c for storage and further analysis. euthanasia criteria was defined as: mouse displays severely hunched posture, inability or reluctance to move, appears weak (staggering when moving around cage), labored breathing, or weight loss of greater than 12% of starting body weight. anti-cchfv-gpc igg elisa development. iodixanol gradient purified δgrvsv-cchfv-gpcδ and rvsv-gfp were re-suspended in np-40 buffer and bca protein quantified (previously described above) and were used as whole virion antigens in coating immunosorbent 96-well plates (thermo fisher scientific). matrices of various antigen, blocking, primary antibodies (hyperimmune mouse ascetic fluid [hmaf kindly provided by t. ksiazek], 8a1, and 11e7) to cchfv/cchfv-gpc, and secondary antibody (mab hrp-goat-α-mouse) concentrations were used to develop optimal detection conditions for the cchfv-gpc via elisa. per optimizations, one microgram of purified antigen per ml was suspended in sterile filtered sodium bicarb/carbonate buffer (ph 9.6) and allowed to incubate on immunosorbent plates overnight at 4 °c. plates were washed with pbs containing a concentration 0.1% tween-20 and 0.001% thimerosal. blocking occurred with 5% milk dissolved in wash buffer, for 2 hrs at room temperature. sera from stat-1 −/− mice was added 1:100 and diluted 2-fold by pipetting across plates and allowed to incubate for one hr at 37 °c. plates were washed and a secondary anti-mouse antibody conjugated to hrp was added at 1:5,000 dilution for one hr at 37 °c. abts peroxidase substrate (kpl, seracare life sciences, milford, ma) was incubated for 15 mins at room temperature prior to the addition of a 1% sds stop solution. plates were read with nine reads per well at 405 nm with a plastic correction factor accounted for from a 490 nm reading per well. test sera was evaluated using both purified δgrvsv-cchfv-gpcδ and rvsv-gfp antigen. plaque reduction neutralization assay. serial dilutions of sera from four mice per treatment group, were aliquoted into cluster tubes with d10 and allowed to incubate with 100 pfu of cchfv, isolate turkey 2004, for approximately 2 hrs on ice. resulting sera plus virus mixture was then overlaid onto 6-well plates of confluent sw-13-cdc cells and absorbed for 1 hr at 37 °c with 5% co 2 with rocking at 15 min intervals. plaque assays were carried out in a manner described above in previous methods section. resulting plaques were enumerated from virus + sera wells and compared to sera plus media only wells run for each sample, and a percent neutralization was calculated and reported for each dilution. hyperimmune mouse ascitic fluid [hmaf] raised against cchfv was additionally serially diluted and run as a positive control. immunohistochemistry of tissues. tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. slides went through heat antigen retrieval in a steamer at 95 °c for 20 mins in sigma citrate buffer, ph6.0, 10× (sigma aldrich, st. louis, mo). to block endogenous peroxidase activity, slides were treated with a 3% hydrogen peroxide and rinsed in distilled water. the tissue sections were processed for ihc using the thermo autostainer 360 (thermofisher, kalamazoo, mi). sequential 15 min incubations with avidin d and biotin solutions (vector, burlingame, ca) were performed to block endogenous biotin reactivity. specific anti-cchfv immunoreactivity was detected using a primary polyclonal rabbit-α-cchfv-np antibody (ibt bioservices, rockville, md) at a 1:3200 dilution for 60 mins. a secondary biotinylated goat-α-rabbit-igg (vector laboratories, burlingame, ca) at 1:200 dilution for 30 mins followed by vector horseradish peroxidase streptavidin, r.t.u (vector) for 30 mins. slides were developed with dako dab chromagen (dako, carpenteria, ca) for 5 mins and counterstained with harris hematoxylin for 30 seconds. tissue sections from uninfected mice were used as negative controls. changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the role of ticks in the maintenance and transmission of crimean-congo hemorrhagic fever virus: a review of published field and laboratory studies crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity crimean-congo haemorrhagic fever crimean-congo hemorrhagic fever virus: new outbreaks, new discoveries treatment of crimean-congo hemorrhagic fever characterization of the glycoproteins of crimean-congo hemorrhagic fever virus crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by 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virus crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice lethal crimean-congo hemorrhagic fever virus infection in interferon α/β receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model a dna vaccine for crimean-congo hemorrhagic fever protects against disease and death in two lethal mouse models immunization of knock-out α/β interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine mice orally immunized with a transgenic plant expressing the glycoprotein of crimean-congo hemorrhagic fever virus a novel vaccine against crimean-congo haemorrhagic fever protects 100% of animals against lethal challenge in a mouse model crimean-congo hemorrhagic fever virus subunit vaccines induce high levels of neutralizing antibodies but no protection in stat1 knockout mice. vector-borne zoonotic dis immunization with dna plasmids coding for crimean-congo hemorrhagic fever virus capsid and envelope proteins and/or virus-like particles induces protection and survival in challenged mice a crimean-congo hemorrhagic fever (cchf) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with andes virus single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease vesicular stomatitis virus-based vaccines protect nonhuman primates against bundibugyo ebolavirus vesicular stomatitis virus: re-inventing the bullet durability of a vesicular stomatitis virus-based marburg virus vaccine in nonhuman primates vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates six-month safety data of recombinant vesicular stomatitis virus-zaire ebola virus envelope glycoprotein vaccine in a phase 3 double-blind, placebo-controlled randomized study in healthy adults first-in-human evaluation of the safety and immunogenicity of a recombinant vesicular stomatitis virus human immunodeficiency virus-1 vaccine (hvtn 090) efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffi t!) the effect of dose on the safety and immunogenicity of the vsv ebola candidate vaccine: a randomised doubleblind, placebo-controlled phase 1/2 trial clinical development of a recombinant ebola vaccine in the midst of an unprecedented epidemic recombinant vesicular stomatitis viruses from generation of vsv pseudotypes using recombinant δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines crimean-congo hemorrhagic fever in humanized mice reveals glial cells as primary targets of neurological infection analysis of the entry mechanism of crimean-congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system crimean-congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires escrt regulators microtubule-dependent and microtubule-independent steps in crimean-congo hemorrhagic fever virus replication cycle passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the golgi apparatus en route to the plasma membrane identification of two additional translation products from the matrix (m) gene that contribute to vesicular stomatitis virus cytopathology the cytoplasmic tails of uukuniemi virus (bunyaviridae) g(n) and g(c) glycoproteins are important for intracellular targeting and the budding of virus-like particles role of the cytoplasmic tail domains of bunyamwera orthobunyavirus glycoproteins gn and gc in virus assembly and morphogenesis vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein identification of a putative crimean-congo hemorrhagic fever virus entry factor proteomics computational analyses suggest that the carboxyl terminal glycoproteins of bunyaviruses are class ii viral fusion protein (beta-penetrenes) identification of human linear b-cell epitope sites on the envelope glycoproteins of crimean-congo haemorrhagic fever virus epitope-mapping of the glycoprotein from crimean-congo hemorrhagic fever virus using a microarray approach murine infection by vesicular stomatitis virus: initial characterization of the h-2d system neurovirulence mutant of vesicular stomatitis virus with an altered target cell tropism in vivo stat1-deficient mice are not an appropriate model for efficacy testing of recombinant vesicular stomatitis virus-based filovirus vaccines heat shock protein 70 family members interact with crimean-congo hemorrhagic fever virus and hazara virus nucleocapsid proteins and perform a functional role in the nairovirus replication cycle immunogenicity of combination dna vaccines for rift valley fever virus, tick-borne encephalitis virus, hantaan virus, and crimean congo hemorrhagic fever virus a cynomolgus macaque model for crimean-congo haemorrhagic fever single-dose attenuated vesiculovax vaccines protect primates against ebola makona virus statistical analysis. a kaplan-meier survival curve was constructed with graphpad prism software (graphpad software, inc., san diego, ca). a power analysis utilizing parameters that included survival, neutralization capacity, and elisa antibody titers from previous pilot studies was performed to assess adequate animal numbers for the current study to differentiate between surviving groups versus control groups to obtain a statistical significance of p < 0.05 with at least a 90% probability. the authors would like to thank the utmb molecular genomics core, assay development core (for deep sequencing support), animal resource center (for handling and husbandry of laboratory animals), electron microscopy laboratory core (specifically, vsevolod popov and julie wen for electron microscopy support), and natalie dobias for immunohistochemistry support. we gratefully acknowledge krystle agans, katharina schmitz, gordon wong, auja smith, angel padilla, emily mantlo, teresa sorvillo, courtney woolsey, corey fulton, stephanie foster, maria cajimat, benjamin satterfield, and daniel deer, viktoriya borisevich, joan geisbert, for various in vitro and in vivo technical guidance and support throughout the project. we thank thomas ksiazek, michael whitt, jeroen kortekaas, and éric bergeron for the gifts of vector/insert plasmids, reagents, cell lines, and virus strains. additionally, we further thank éric bergeron for fruitful discussions and unpublished data regarding cchfv-gpc reagents and thomas ksiazek for elisa development discussions. this research was conducted by s.e.r. in partial fulfillment of the requirements for a ph.d. from the university of texas medical branch, galveston, texas, usa. funding and support was provided by the utmb department of microbiology and immunology to t.w.g. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44210-6.competing interests: s.e.r., r.w.c., c.e.m. and t.w.g. have filed a provisional patent application for replication competent vesicular stomatitis vector encoding crimean-congo hemorrhagic fever antigen.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-011435-x73foqu7 authors: glanz, anna; chawla, karan; fabry, stephanie; subramanian, gayatri; garcia, julie; jay, bryanna; ciricillo, jacob; chakravarti, ritu; taylor, r. travis; chattopadhyay, saurabh title: high throughput screening of fda-approved drug library reveals the compounds that promote irf3-mediated pro-apoptotic pathway inhibit virus replication date: 2020-04-14 journal: viruses doi: 10.3390/v12040442 sha: doc_id: 11435 cord_uid: x73foqu7 interferon (ifn) regulatory factor 3 (irf3) is the key transcription factor for the induction of ifn and antiviral genes. the absence of antiviral genes in irf3 deficiency leads to susceptibility to a wide range of viral infections. previously, we uncovered a function for nontranscriptional irf3 (nt-irf3), rlr (rig-i-like receptor)-induced irf3-mediated pathway of apoptosis (ripa), which triggers apoptotic killing of virus-infected cells. using knock-in mice expressing a transcriptionally inactive, but ripa-active, irf3 mutant, we demonstrated the relative contribution of ripa to host antiviral defense. given that ripa is a cellular antiviral pathway, we hypothesized that small molecules that promote ripa in virus-infected cells would act as antiviral agents. to test this, we conducted a high throughput screen of a library of fda-approved drugs to identify novel ripa activators. our screen identified doxorubicin as a potent ripa-activating agent. in support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate irf3 in ripa. surprisingly, doxorubicin inhibited the transcriptional activity of irf3. the antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate irf3. mechanistically, doxorubicin promoted ripa by activating the extracellular signal-regulated kinase (erk) signaling pathway. finally, we validated these results using another ripa-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of irf3. therefore, we demonstrate that the ripa branch of irf3 can be targeted therapeutically to prevent virus infection. the innate immune response is the first line of defense against microbial infection. the interferon (ifn) system represents a key antiviral innate immune response mechanism that dictates the outcome of a viral infection [1] . ifn-β, a type-i ifn, is synthesized in the virus-infected cells by the transcriptional activity of ifn regulatory factor 3 (irf3) [2, 3] . irf3 remains as an inactive monomer in the cytosol of the uninfected cells; upon virus infection, it gets phosphorylated, dimerized, and translocated to the nucleus [2, 4] . in the nucleus, the dimeric irf3 binds to the promoters of ifn-β and many ifn-stimulated genes (isgs). secreted ifns act via autocrine and paracrine signaling to amplify the transcriptional induction of hundreds of isgs [5] . the isg-encoded protein products act on specific stages of the virus life cycle to inhibit viral replication and pathogenesis. the absence of antiviral genes in irf3 −/− mice causes high susceptibility to a wide range of viruses [6, 7] . we have uncovered that, in addition to the transcriptional activity, irf3 functions in a nontranscriptional (nt) pathway in antiviral defense [8] [9] [10] [11] [12] [13] [14] [15] . in contrast to the transcriptional pathway, nt-irf3 in virus-infected cells functions as a chaperone protein by translocating the pro-apoptotic protein bcl2-associated x (bax) to the mitochondria, thereby causing apoptotic cell death, which we named rlr (rig-i-like receptor)-induced irf3-mediated pathway of apoptosis (ripa) ( figure 1a ) [7] [8] [9] [10] [11] [12] [13] . in ripa, irf3 is activated by linear ubiquitination on two key lysine residues by linear ubiquitin chain assembly complex (lubac) to allow its interaction with the pro-apoptotic protein bax [7, 12] . the irf3/bax complex translocates to the mitochondrial membrane and stimulates the release of cytochrome c into the cytosol. cytosolic cytochrome c activates cellular caspases to trigger apoptotic cell death ( figure 1a ). recently, we demonstrated that ripa contributes to the optimal antiviral activity of irf3. knock-in mice expressing a ripa-active nt-irf3 mutant (irf3 s1/s1 ) can mount antiviral protection against respiratory pathogenesis by sendai virus (sev) [7] . the sev-infected cells, in the absence of ripa, establish viral persistence [14] . sev temporarily regulates ripa by activating the cellular survival pathways, e.g., the phosphatidylinositol 3-kinase (pi3k)-dependent akt, which stabilizes x-linked inhibitor of apoptosis protein (xiap), an inhibitor of ripa [15] . the inhibition of pi3k rapidly triggers ripa to kill the virus-infected cells [15] . in the time since we described a function for nt-irf3, several studies have reported ripa-like activities in viral and nonviral pathogenesis. in human t cell leukemia virus (htlv1)-infected primary monocytes, stimulator of interferon genes (sting)-activated irf3 interacts with bax to cause apoptosis. the irf3/bax-mediated monocyte cell death prevents productive htlv1 replication [16] . in hepatocytes, sting-activated irf3 causes alcoholic liver diseases (ald) during chronic ethanol administration in mice [17] . ethanol administration triggers endoplasmic reticulum stress, which activates sting signaling to enable an interaction between irf3 and bax, leading to hepatocyte apoptosis. a subsequent study further revealed that carbon tetrachloride (ccl4)-induced hepatotoxicity is caused by the ripa-like activity of irf3, mediated by sting/irf3/bax-dependent apoptotic pathway. to further investigate the role of ripa in ald, we used the irf3 s1/s1 knock-in mice in a mouse alcoholic hepatitis model to show that ethanol administration activates ripa in hepatic immune cells. since the immune cells are necessary for the resolution of liver injury, our study demonstrated a detrimental role for ripa in ald pathogenesis [18] . in contrast, irf3 s1/s1 mice are protected in high-fat diet (hfd)-induced liver diseases by the resolution of hepatic inflammation [19] . the involvement of ripa in various disease models highlights its potential as a therapeutic target. to test this, we took a pharmacological approach to isolate small molecule modifiers of ripa. in the current study, we performed a high throughput screen of a library of fda-approved compounds (prestwick chemical), and isolated a small subset of ripa-promoting compounds. using two compounds, which specifically activated ripa, but not the transcriptional function of irf3, we demonstrated that therapeutic activation of the ripa branch of irf3 inhibits virus replication. human cell lines mda-mb-453 (atcc htb-131), ht1080 (atcc ccl-121), and a549 (atcc ccl-185), the african green monkey cell line vero (atcc ccl-81), and mouse embryonic fibroblasts (mefs) were maintained in dmem containing 10% fbs, penicillin, and streptomycin. all cell lines viruses 2020, 12, 442 3 of 18 used in this study were maintained in the authors' laboratory. expression vectors of human irf3 and irf3-k10 were described previously [7] , and the ligands for retinoic acid-inducible gene-i (rig-i), toll-like receptor 3 (tlr3), and sting have been described before [7, 20] . the fda-approved drug library was obtained from prestwick chemical (pc, washington, dc, usa). individual chemicals were obtained from sigma-aldrich ( , anti-ifit1 (described previously [7, 9] ), anti-ifit3 (described previously [7, 9] ), and anti-vsv g-protein (described previously [21, 22] ). the human breast cancer cell line mda-mb-453 was used to screen the library of fda-approved drugs to isolate the regulators of ripa ( figure 1f ). the cells were seeded in 96-well black-bottom tissue culture plates, and the next day, the cells were transfected with polyi:c using lipofectamine 2000 to stimulate ripa. after the addition of the transfection complex, the cells were immediately treated with either dmso (vehicle) or the drug library (at 20 µm final concentration). after 20 h of the ripa stimulation, the cells were analyzed for caspase activity using apo-onetm homogeneous caspase-3/7 assay (promega, san luis obispo, ca, usa) following the manufacturer's instructions. the caspase activity of the vehicle-treated well was arbitrarily considered as 100 and all other values were normalized to this. a representative drug screening plate is shown in figure s1 . the normalized caspase activity was used to calculate the z-scores and the top 25 primary hits were isolated for further validation. ripa stimulation was performed by transfecting the cells with polyi:c (1 µg if not indicated otherwise in the figure legends) using lipofectamine 2000 for 16-24 h, and the cells were either analyzed for caspase activity or cleaved parp, as indicated in the figure legends. for the drug treatments, the cells were pretreated with the drugs (e.g., doxorubicin at 5 µm, and pyrvinium pamoate at 1 µm or as indicated in the figures) for 1 h before ripa stimulation (polyi:c transfection). dmso was used as a vehicle control for these drugs. ht1080 cells were transfected with either control (sc-418922) or irf3-specific (sc-417171) crispr/cas9 plasmids (santa cruz) using lipofectamine 2000 (thermo fisher scientific). transfected cells were sorted for high gfp-expressers using flow cytometry, and the gfp-expressing cells were expanded to isolate individual clones. these clones were screened for irf3 protein levels by immunoblot, and the clones with no irf3 protein expression were expanded and further validated using functional assay to ensure the absence of irf3-dependent ifit3 induction upon rlr stimulation ( figure s2 ). vesicular stomatitis virus (vsv) indiana strain expressing green fluorescent protein (gfp), sendai virus (sev) cantell strain (charles river laboratories, garfield heights, oh, usa), herpes simplex virus 1 (hsv-1) kos and f strains, langat virus (lgtv), kunjin virus (kunv), and the infection procedures have been described previously [13, 20, 23] . briefly, the cells were infected with the viruses [at a multiplicity of infection (moi) of 1] in serum-free dmem for 2 h, after which the cells were washed and replaced with normal growth medium. the virus-infected cells were analyzed at the indicated time for viral protein expression or as described in figure legends. for quantification of infectious virus particles in the culture medium, plaque assays were performed for vsv in 10-fold serial dilution on vero cells [24] . hsv-1 titer was measured by tcid50 using 10-fold serial dilution of the culture media on vero cells [25] . plaque assays were performed for lgtv and kunv using previously described procedures [23] . to determine the effects of the drugs on viral replication, the cells were pre-treated with dmem containing dmso (vehicle) or individual drugs at the indicated concentrations for 2 h before virus infection, removed during the virus adsorption, and reintroduced post adsorption. equal protein extracts from the gfp.vsv-infected cells were analyzed for gfp fluorescence using a plate reader. gfp fluorescence of vehicle-treated vsv-infected cells was set arbitrarily at 100 and all other values were normalized to this. immunoblot analyses were performed using previously described procedures [7, 9] . briefly, the cells were lysed in 50 mm tris buffer, ph 7.4, containing 150 mm of nacl, 0.1% triton x-100, 1 mm sodium orthovanadate, 10 mm of sodium fluoride, 10 mm of β-glycerophosphate, 5 mm sodium pyrophosphate, protease and phosphatase inhibitors (roche). total protein extracts were analyzed by sds-page followed by immunoblot. for analyzing the ubiquitination of irf3, a previously described procedure was followed [7] . the immunoblots were quantified by image j software. total rna was isolated using trizol (thermo fisher scientific), cdna was prepared using improm-ii reverse transcription kit (promega), and the cdna was analyzed using radianttm sybr green pcr mix (alkali scientific inc., fort lauderdale, fl, usa) in roche lightcycler 96 instrument and analyzed with the lightcycler 480 software, version 1.5. the expression levels of the mrnas were normalized to 18s rrna. for the qrt-pcr analyses of the respective genes, the following primers were used: ifit1-fwd: tctcagaggagcctggctaag, ifit1-rev: gtcaccagactcctcacatttgc, ifit3-fwd: gaacatgctgaccaagcaga, ifit3-rev: cagttgtgtccacccttcct, ifnb1-fwd: cgccgcattgaccatcta, ifnb1-rev: gacattagccaggaggttct, 18s-fwd: attgacggaagggcaccaccag, 18s-rev: caaatcgctccaccaactaagaacg. mda-mb-453 cells were grown on coverslips, infected with hsv-1 in the absence or the presence of doxorubicin, as described in the figure legends. the infected cells were fixed in 4% paraformaldehyde (electron microscopy sciences, hatfield, pa, usa, #15710), permeabilized in 0.2% triton x-100 (fisher scientific #9002-93-1) and immunostained with anti-icp0 antibody followed by alexa. fluor-conjugated secondary antibody (invitrogen #a-11004). the coverslips were mounted on microscopy slides using vectashield/dapi (vector laboratories, burlingame, ca, usa, #h-1200) and analyzed using an olympus (waltham, ma, usa) confocal microscope and olympus fluoview fv1000 software. the caspase-3/7 activity of the cell lysates was performed using previously described procedures [9] . briefly, the cell lysates were used for measuring caspase activity using the apo-onetm homogeneous caspase-3/7 assay according to protocols provided by the manufacturer (promega). caspase activity in experimental samples was plotted relative to rlr-stimulated cells arbitrarily set as 100 (as in figure 1d ). cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay, trypan blue exclusion, and brightfield microscopy. to measure cell viability, the cells were seeded in a 96-well plate, and transfected (rlr) or treated (tlr3) with polyi:c in the absence or the presence of doxorubicin or vehicle (dmso), as indicated, for 24 h. the mtt assay was performed using previously described procedures [26] . the absorbance of the treated cells was considered as 100, and the other values were normalized to this. for the trypan blue exclusion assay, the treated cells were trypsinized, resuspended in complete dmem, stained with trypan blue and the live and dead cells were counted using a hemocytometer to calculate percent viability for each treatment. the statistical analyses were performed using graphpad prism 5.02 software. the "p" values were calculated using two-tailed, unpaired student's t-tests and are shown in the relevant figures. the results presented here are the representatives of at least three biological repeats. to identify small molecule activators of the antiviral ripa branch of irf3 ( figure 1a ), we performed an unbiased high throughput screen using a library of fda-approved compounds. activation of ripa, by transfection of the rlr ligand polyi:c, caused robust cell death ( figure 1b , stage-5, figure 1a ). stimulation of tlr3 or sting, by their cognate ligands, did not cause visible cell death ( figure 1b ). cleaved parp (c-parp, stage-4, figure 1a ), a molecular marker of apoptosis, was observed in rlr-, but not sting-stimulated, cells. as expected, the apoptotic executioner caspase, caspase-3 (stage-3, figure 1a ), was strongly activated only upon rlr, but not tlr3 or sting, stimulation for an increasing time period ( figure 1d ,e). therefore, we optimized conditions to screen for agents that specifically modulate ripa-induced apoptosis, without the need to separate the contribution of tlr3 or sting pathways. to isolate the activators of ripa, we performed a high throughput screen of a library consisting of 1200 fda-approved compounds [27] . the library is 96-well formatted with each well containing an individual compound. we performed the primary screen using the strategy outlined in figure 1f , and the activity of caspase-3 was used as a readout of the ripa activity in each well. the caspase-3 activity of vehicle (dmso)-treated well was arbitrarily set at 100, and all other values were normalized to this (a sample plate is shown in figure s1 ). the primary screen resulted in several ripa activators, and using an arbitrary cut-off of z-scores greater than 1.15, we obtained twenty-five candidate ripa activating compounds (figure 2a ,b, table s1 ). we noted that the ripa activators constitute only 2% of the library, indicating the specificities of the compounds. the primary hits were validated using a secondary screen, and the ripa-promoting activity of each compound was determined with respect to the vehicle control. the secondary screen validated twenty-four of the twenty-five compounds obtained from the primary screen ( figure 2c ). the secondary-validated ripa-activators consisted of compounds from a variety of therapeutic activities, e.g., anticancer, antibacterial, anti-inflammatory, antihypertensive, etc ( figure 2b ). we excluded act1, which triggered caspase-3 activity nonspecifically, from the subsequent studies, and focused on doxorubicin, which has earlier been studied to activate irf3 [28] . because ripa contributes to the antiviral function of irf3 [7] , we hypothesized that agents that promote ripa would inhibit viral replication. for this purpose, we used vesicular stomatitis virus (vsv) as a model negative-sense rna virus. doxorubicin treatment inhibited vsv replication, analyzed by virus-encoded gfp expression ( figure 3a ). we validated these results by immunoblot analyses, which demonstrate that doxorubicin treatment inhibited the expression of vsv-g protein, a viral envelope glycoprotein as well as the virus-encoded gfp ( figure 3b ). as expected, the reduced viral protein expression led to reduced infectious virus particle release from doxorubicin-treated cells ( figure 3c ). we validated the results in another cell line (a549), in which doxorubicin also inhibited vsv-encoded gfp expression ( figure 3d ). to determine whether the antiviral activity of doxorubicin is dependent on irf3, we generated irf3 −/− human cells (ht1080) using a crispr/cas9 approach ( figure 3e , lower panel, figure s2 ). doxorubicin, as expected, inhibited the viral protein expression in the wt cells; however, the antiviral activity of doxorubicin was impaired in irf3 −/− cells ( figure 3e , top panel). to further validate this striking result, we used wt and irf3 −/− mouse embryonic fibroblasts (mefs) ( figure 3f , lower panel). similar to the human cells, doxorubicin inhibited vsv replication only in the presence of irf3 ( figure 3f , top panel). we further quantified, in addition to viral g protein, the expression of vsv-encoded gfp fluorescence. similar to the inhibition of g protein expression, doxorubicin inhibited gfp fluorescence in wt but not irf3-deficient cells ( figure 3e , f). we ensured that doxorubicin did not cause substantial cytotoxicity under our experimental conditions ( figure s3a , b). our results demonstrate, for the first time, that doxorubicin is a ripa-promoting agent, which inhibits viral infection in an irf3-dependent manner. because ripa contributes to the antiviral function of irf3 [7] , we hypothesized that agents that promote ripa would inhibit viral replication. for this purpose, we used vesicular stomatitis virus (vsv) as a model negative-sense rna virus. doxorubicin treatment inhibited vsv replication, analyzed by virus-encoded gfp expression ( figure 3a ). we validated these results by immunoblot analyses, which demonstrate that doxorubicin treatment inhibited the expression of vsv-g protein, a viral envelope glycoprotein as well as the virus-encoded gfp ( figure 3b ). as expected, the reduced viral protein expression led to reduced infectious virus particle release from doxorubicin-treated cells ( figure 3c ). we validated the results in another cell line (a549), in which doxorubicin also inhibited vsv-encoded gfp expression ( figure 3d ). to determine whether the antiviral activity of doxorubicin is dependent on irf3, we generated irf3 −/− human cells (ht1080) using a crispr/cas9 approach ( figure 3e , lower panel, figure s2 ). doxorubicin, as expected, inhibited the viral protein expression in the wt cells; however, the antiviral activity of doxorubicin was impaired in irf3 −/− cells ( figure 3e , top panel). to further validate this striking result, we used wt and irf3 −/− mouse embryonic fibroblasts (mefs) ( figure 3f , lower panel). similar to the human cells, doxorubicin inhibited vsv replication only in the presence of irf3 ( figure 3f, top panel) . we further quantified, in addition to viral g protein, the expression of vsv-encoded gfp fluorescence. similar to the inhibition of g protein expression, doxorubicin inhibited gfp fluorescence in wt but not irf3-deficient cells ( figure 3e ,f). we ensured that doxorubicin did not cause substantial cytotoxicity under our experimental conditions ( figure s3a ,b). our results demonstrate, for the first time, that doxorubicin is a ripa-promoting agent, which inhibits viral infection in an irf3-dependent manner. the optimal antiviral action of irf3 depends on its transcriptional and nontranscriptional (nt, ripa) functions. we sought to determine whether doxorubicin activates either one or both functions of irf3. furthermore, doxorubicin has previously been reported to induce phosphorylation and transcriptional activation of irf3 [28] . thus, we examined whether doxorubicin promotes the transcriptional activity of irf3 by rlr-signaling in vsv-infected cells. vsv infection induced irf3target gene, ifit3, which was strongly inhibited by doxorubicin, examined at multiple doses ( figure 4a ). in contrast, doxorubicin robustly enhanced the ripa activity, i.e., the c-parp levels in vsvinfected cells ( figure 4a ). to solidify these results, we used a nonviral rlr agonist, polyi:c, and the irf3-induced ifit3 was also strongly inhibited by doxorubicin in ht1080 cells ( figure 4b ). similar to vsv-infected cells, doxorubicin promoted irf3-induced ripa activity in these cells (c-parp, figure 4b ). to validate these results, we measured the transcriptional induction of additional irf3induced genes, both at the protein and mrna levels. the irf3-induced ifit3 and ifit1 proteins were strongly inhibited by doxorubicin at multiple times post rlr-stimulation ( figure 4c ). doxorubicin treatment also significantly inhibited the mrna induction of ifit3 ( figure 4d ), ifit1 ( figure 4e ), and ifn-β ( figure 4f ), all of which depend on the transcriptional activity of irf3. collectively, our results demonstrate that doxorubicin inhibits vsv replication in the absence of irf3induced antiviral gene expression. the optimal antiviral action of irf3 depends on its transcriptional and nontranscriptional (nt, ripa) functions. we sought to determine whether doxorubicin activates either one or both functions of irf3. furthermore, doxorubicin has previously been reported to induce phosphorylation and transcriptional activation of irf3 [28] . thus, we examined whether doxorubicin promotes the transcriptional activity of irf3 by rlr-signaling in vsv-infected cells. vsv infection induced irf3-target gene, ifit3, which was strongly inhibited by doxorubicin, examined at multiple doses ( figure 4a ). in contrast, doxorubicin robustly enhanced the ripa activity, i.e., the c-parp levels in vsv-infected cells ( figure 4a ). to solidify these results, we used a nonviral rlr agonist, polyi:c, and the irf3-induced ifit3 was also strongly inhibited by doxorubicin in ht1080 cells ( figure 4b ). similar to vsv-infected cells, doxorubicin promoted irf3-induced ripa activity in these cells (c-parp, figure 4b ). to validate these results, we measured the transcriptional induction of additional irf3-induced genes, both at the protein and mrna levels. the irf3-induced ifit3 and ifit1 proteins were strongly inhibited by doxorubicin at multiple times post rlr-stimulation ( figure 4c ). doxorubicin treatment also significantly inhibited the mrna induction of ifit3 ( figure 4d ), ifit1 ( figure 4e) , and ifn-β ( figure 4f ), all of which depend on the transcriptional activity of irf3. collectively, our results demonstrate that doxorubicin inhibits vsv replication in the absence of irf3-induced antiviral gene expression. doxorubicin promoted the ripa activity, measured by robust increase in rlr-induced c-parp levels, when compared with rlr-treated cells ( figure 5a,b) . doxorubicin is a known inducer of the cellular apoptotic pathway [29, 30] , and, as expected, also triggered apoptosis in the absence of rlr stimulation ( figure 5a , lane 2). to inquire genetically how doxorubicin activates irf3 to promote ripa, we took a strategy to measure the "doxorubicin-promoted ripa activity" using the combined treatment of rlr and doxorubicin ("rlr/doxo", lane 4, figure 5a ), to avoid the individual cellular effects of rlr or doxorubicin (lanes 2, 3, figure 5a ). similar to c-parp, doxorubicin significantly increased the rlr-induced caspase-3 activity ( figure 5c ). the increased ripa activity by doxorubicin, as expected, led to enhanced cell death in a dose-dependent manner ( figure s3c ). we examined doxorubicin-promoted ripa in wt and irf3 −/− cells. doxorubicin caused robust increase in rlr-induced c-parp and capsase-3 activation in wt cells (figures 5d, e) . the rlr/doxorubicininduced c-parp and caspase activity were reduced in irf3 −/− cells ( figure 5d , e). because doxorubicin specifically promotes the ripa, and not the transcriptional activity of irf3, we used a nt-irf3 mutant, irf3-k10, which is functional only in the ripa, but not the transcriptional branch [7] . the irf3-k10-expressing cells showed strong promotion of ripa activity upon doxorubicin treatment ( figure 5f ). collectively, our results demonstrate that doxorubicin inhibits the transcriptional, but promotes the ripa, activity of irf3. doxorubicin promoted the ripa activity, measured by robust increase in rlr-induced c-parp levels, when compared with rlr-treated cells ( figure 5a,b) . doxorubicin is a known inducer of the cellular apoptotic pathway [29, 30] , and, as expected, also triggered apoptosis in the absence of rlr stimulation ( figure 5a , lane 2). to inquire genetically how doxorubicin activates irf3 to promote ripa, we took a strategy to measure the "doxorubicin-promoted ripa activity" using the combined treatment of rlr and doxorubicin ("rlr/doxo", lane 4, figure 5a ), to avoid the individual cellular effects of rlr or doxorubicin (lanes 2, 3, figure 5a ). similar to c-parp, doxorubicin significantly increased the rlr-induced caspase-3 activity ( figure 5c ). the increased ripa activity by doxorubicin, as expected, led to enhanced cell death in a dose-dependent manner ( figure s3c ). we examined doxorubicin-promoted ripa in wt and irf3 −/− cells. doxorubicin caused robust increase in rlr-induced c-parp and capsase-3 activation in wt cells ( figure 5d ,e). the rlr/doxorubicin-induced c-parp and caspase activity were reduced in irf3 −/− cells ( figure 5d ,e). because doxorubicin specifically promotes the ripa, and not the transcriptional activity of irf3, we used a nt-irf3 mutant, irf3-k10, which is functional only in the ripa, but not the transcriptional branch [7] . the irf3-k10-expressing cells showed strong promotion of ripa activity upon doxorubicin treatment ( figure 5f ). collectively, our results demonstrate that doxorubicin inhibits the transcriptional, but promotes the ripa, activity of irf3. to investigate the biochemical mechanism by which doxorubicin promotes ripa, we examined whether doxorubicin activates any specific step(s) of ripa (depicted in figure 1a ). we systematically analyzed both pre-and postmitochondrial steps of ripa. the ubiquitination of irf3 (stage-1, figure 1a ), which is essential for activating ripa, was not promoted by doxorubicin ( figure 6a ). we noted that doxorubicin-treated cells exhibited shorter ubiquitin chains linked to irf3 than the vehicletreated cells. the results led us to examine whether doxorubicin increased the rlr-induced release of cytochrome c into the cytosol (stage-2, figure 1a ), a step that requires the interaction of irf3 and bax. doxorubicin did not increase the rlr-induced release of cytosolic cytochrome c ( figure 6b ). these results led us to hypothesize that doxorubicin activates rlr-activated cellular signaling to promote ripa. a previous study indicated that p53, which can be activated by doxorubicin [24] , is not involved in rlr-induced apoptosis [31] . therefore, we examined the c-jun n-terminal kinase (jnk) and extracellular signal-regulated kinase (erk) signaling pathways, which are activated upon rlr stimulation [32, 33] . rlr stimulation triggered the phosphorylation of jnk (pjnk) and erk (perk), which were enhanced by doxorubicin ( figure 6c, d) . we then tested whether doxorubicinactivated jnk and erk are involved in promoting ripa, using pharmacological inhibitors of jnk and mitogen-activated protein kinase kinase (mek), the upstream activator of erk. inhibition of mek (u0126, meki), but not jnk (sp600125, jnki), significantly suppressed doxorubicin-promoted ripa ( figure 6e ). we further validated the role of erk in mouse cells expressing the nt-irf3 mutant (irf3 s1/s1 ), which is active in ripa [7] . in irf3 s1/s1 mefs, the mek inhibitor suppressed doxorubicin to investigate the biochemical mechanism by which doxorubicin promotes ripa, we examined whether doxorubicin activates any specific step(s) of ripa (depicted in figure 1a ). we systematically analyzed both pre-and postmitochondrial steps of ripa. the ubiquitination of irf3 (stage-1, figure 1a) , which is essential for activating ripa, was not promoted by doxorubicin ( figure 6a ). we noted that doxorubicin-treated cells exhibited shorter ubiquitin chains linked to irf3 than the vehicle-treated cells. the results led us to examine whether doxorubicin increased the rlr-induced release of cytochrome c into the cytosol (stage-2, figure 1a ), a step that requires the interaction of irf3 and bax. doxorubicin did not increase the rlr-induced release of cytosolic cytochrome c ( figure 6b ). these results led us to hypothesize that doxorubicin activates rlr-activated cellular signaling to promote ripa. a previous study indicated that p53, which can be activated by doxorubicin [24] , is not involved in rlr-induced apoptosis [31] . therefore, we examined the c-jun n-terminal kinase (jnk) and extracellular signal-regulated kinase (erk) signaling pathways, which are activated upon rlr stimulation [32, 33] . rlr stimulation triggered the phosphorylation of jnk (pjnk) and erk (perk), which were enhanced by doxorubicin ( figure 6c,d) . we then tested whether doxorubicin-activated jnk and erk are involved in promoting ripa, using pharmacological inhibitors of jnk and mitogen-activated protein kinase kinase (mek), the upstream activator of erk. inhibition of mek (u0126, mek i ), but not jnk (sp600125, jnk i ), significantly suppressed doxorubicin-promoted ripa ( figure 6e ). we further validated the role of erk in mouse cells expressing the nt-irf3 mutant (irf3 s1/s1 ), which is active in ripa [7] . in irf3 s1/s1 mefs, the mek inhibitor suppressed doxorubicin-promoted ripa, analyzed by rlr-induced caspase-3 activity ( figure 6f ) and c-parp ( figure 6g ). together, doxorubicin-activated erk signaling is involved in promoting ripa. viruses 2020, 12, x for peer review 11 of 18 promoted ripa, analyzed by rlr-induced caspase-3 activity ( figure 6f ) and c-parp ( figure 6g ). together, doxorubicin-activated erk signaling is involved in promoting ripa. to test whether doxorubicin inhibits viruses of other families, we used members of flaviviridae and herpesviridae, which are known to activate the rlr signaling pathway [34] [35] [36] . we previously demonstrated that ripa can be activated by viruses with positive-sense rna or dna genomes [13] . consistent with the vsv results (figure 3) , doxorubicin treatment inhibited langat virus (lgtv) ( figure 7a) and kunjin virus (kunv) ( figure 7b ) infectious virion production. furthermore, cytosolic dna from dna viruses can activate ripa through rna polymerase iii activity [13] . to investigate whether doxorubicin also inhibits dna virus replication, we used two strains (kos and f) of hsv-1. doxorubicin strongly inhibited hsv-1 (kos strain) replication, indicated by the reduction in icp0 expression by immunoblot ( figure 7c ) and confocal microscopy ( figure 7d ). the inhibition of viral protein expression led to a significant reduction in infectious virus particle release from the doxorubicin-treated cells ( figure 7e ). similar to the kos strain, doxorubicin also inhibited the replication of hsv-1 f strain ( figure 7f ). collectively, our results demonstrate that the ripapromoting agent doxorubicin inhibits the replication of both rna and dna viruses. to test whether doxorubicin inhibits viruses of other families, we used members of flaviviridae and herpesviridae, which are known to activate the rlr signaling pathway [34] [35] [36] . we previously demonstrated that ripa can be activated by viruses with positive-sense rna or dna genomes [13] . consistent with the vsv results (figure 3) , doxorubicin treatment inhibited langat virus (lgtv) ( figure 7a) and kunjin virus (kunv) ( figure 7b ) infectious virion production. furthermore, cytosolic dna from dna viruses can activate ripa through rna polymerase iii activity [13] . to investigate whether doxorubicin also inhibits dna virus replication, we used two strains (kos and f) of hsv-1. doxorubicin strongly inhibited hsv-1 (kos strain) replication, indicated by the reduction in icp0 expression by immunoblot ( figure 7c ) and confocal microscopy ( figure 7d ). the inhibition of viral protein expression led to a significant reduction in infectious virus particle release from the doxorubicin-treated cells ( figure 7e ). similar to the kos strain, doxorubicin also inhibited the replication of hsv-1 f strain ( figure 7f ). collectively, our results demonstrate that the ripa-promoting agent doxorubicin inhibits the replication of both rna and dna viruses. viruses 2020, 12, x for peer review 12 of 18 to test the generality of the theme that ripa-promoting compounds are antiviral agents, we screened a subset of the secondary-validated ripa-promoters (act12, act2, act5, act16, act9, and act3), based on an arbitrary cut-off of a 1.9-fold increase in ripa-promotion ( figure 2c ), for their ability to inhibit hsv-1 replication ( figure 8a) . we confirmed the anti-hsv-1 activity of doxorubicin (act9), and also isolated two additional compounds (act3 and act5) that strongly inhibited icp0 expression ( figure 8a ). we excluded act3 (topotecan) from further analyses because it shares properties with doxorubicin and may activate similar pathways. we focused on the anti-helminthic drug pyrvinium pamoate (pp, act5) for validation of our hypothesis. similar to doxorubicin, pp inhibited the replication of hsv-1 in ht1080 cells, in a dose-dependent manner, demonstrated by immunoblot of viral proteins icp0 and icp8 ( figure 8b ). pp also inhibited vsv replication, analyzed by immunoblot of the viral protein vsv-g ( figure 8c ) and infectious virion particle release ( figure 8d ). the antiviral activity of pp prompted us to test its ability to activate irf3. pp, as expected, promoted the ripa branch of irf3, measured by caspase activity ( figure 8e ) and c-parp ( figure 8f ). unlike doxorubicin, pp treatment did not trigger an apoptotic pathway in the absence of rlrstimulation ( figure 8f, lane 2) . to investigate whether pp uses a mechanism similar to doxorubicin to promote ripa, we tested the mek inhibitor in pp-promoted ripa. inhibition of mek reduced rlr-induced c-parp in pp-treated cells ( figure 8g) . therefore, erk signaling may be a common to test the generality of the theme that ripa-promoting compounds are antiviral agents, we screened a subset of the secondary-validated ripa-promoters (act12, act2, act5, act16, act9, and act3), based on an arbitrary cut-off of a 1.9-fold increase in ripa-promotion ( figure 2c ), for their ability to inhibit hsv-1 replication ( figure 8a ). we confirmed the anti-hsv-1 activity of doxorubicin (act9), and also isolated two additional compounds (act3 and act5) that strongly inhibited icp0 expression ( figure 8a ). we excluded act3 (topotecan) from further analyses because it shares properties with doxorubicin and may activate similar pathways. we focused on the anti-helminthic drug pyrvinium pamoate (pp, act5) for validation of our hypothesis. similar to doxorubicin, pp inhibited the replication of hsv-1 in ht1080 cells, in a dose-dependent manner, demonstrated by immunoblot of viral proteins icp0 and icp8 ( figure 8b ). pp also inhibited vsv replication, analyzed by immunoblot of the viral protein vsv-g ( figure 8c ) and infectious virion particle release ( figure 8d ). the antiviral activity of pp prompted us to test its ability to activate irf3. pp, as expected, promoted the ripa branch of irf3, measured by caspase activity ( figure 8e ) and c-parp ( figure 8f ). unlike doxorubicin, pp treatment did not trigger an apoptotic pathway in the absence of rlr-stimulation ( figure 8f, lane 2) . to investigate whether pp uses a mechanism similar to doxorubicin to promote ripa, we tested the mek inhibitor in pp-promoted ripa. inhibition of mek reduced rlr-induced c-parp in pp-treated cells ( figure 8g) . therefore, erk signaling may be a common mechanism to therapeutically target ripa in multiple cell types. next, we inquired about the effect of pp on the transcriptional activity of irf3. interestingly, although pp promoted the ripa branch of irf3 ( figure 8e,f) , it had no effect on the transcriptional activity of irf3, demonstrated by qrt-pcr analyses of irf3 target genes ( figure 8h,i) . therefore, in contrast to doxorubicin, which differentially modulates irf3 activity by promoting ripa and inhibiting the irf3 transcriptional branch, pp exclusively promotes ripa without affecting irf3 transcriptional activity. mechanism to therapeutically target ripa in multiple cell types. next, we inquired about the effect of pp on the transcriptional activity of irf3. interestingly, although pp promoted the ripa branch of irf3 ( figure 8e,f) , it had no effect on the transcriptional activity of irf3, demonstrated by qrt-pcr analyses of irf3 target genes ( figure 8h,i) . therefore, in contrast to doxorubicin, which differentially modulates irf3 activity by promoting ripa and inhibiting the irf3 transcriptional branch, pp exclusively promotes ripa without affecting irf3 transcriptional activity. we have previously demonstrated, using genetically manipulated cells and mice, that nt-irf3 function via ripa contributes to the optimal antiviral activity of irf3 [7] [8] [9] [10] 12 ]. in the current study, we sought to identify small molecules that activate ripa and, therefore, can limit virus replication. using a high-throughput screen of a library of fda-approved drugs, we isolated a subset of compounds with diverse therapeutic functions that promoted the activity of ripa. among the ripaactivating compounds, we focused on doxorubicin for mechanistic and functional studies. doxorubicin inhibits the replication of vsv by activating the ripa branch of irf3. our in-depth biochemical studies revealed that doxorubicin inhibited the transcriptional activity of irf3; moreover, doxorubicin promoted ripa by activating the erk signaling pathway. the antiviral function of doxorubicin was expanded to the tick-and mosquito-borne flaviviruses (lgtv and kunv), and hsv-1. finally, we validated our results using pyrvinium pamoate, which also inhibited the replication of vsv and hsv-1 by specifically promoting ripa, and not the transcriptional, we have previously demonstrated, using genetically manipulated cells and mice, that nt-irf3 function via ripa contributes to the optimal antiviral activity of irf3 [7] [8] [9] [10] 12 ]. in the current study, we sought to identify small molecules that activate ripa and, therefore, can limit virus replication. using a high-throughput screen of a library of fda-approved drugs, we isolated a subset of compounds with diverse therapeutic functions that promoted the activity of ripa. among the ripa-activating compounds, we focused on doxorubicin for mechanistic and functional studies. doxorubicin inhibits the replication of vsv by activating the ripa branch of irf3. our in-depth biochemical studies revealed that doxorubicin inhibited the transcriptional activity of irf3; moreover, doxorubicin promoted ripa by activating the erk signaling pathway. the antiviral function of doxorubicin was expanded to the tick-and mosquito-borne flaviviruses (lgtv and kunv), and hsv-1. finally, we validated our results using pyrvinium pamoate, which also inhibited the replication of vsv and hsv-1 by specifically promoting ripa, and not the transcriptional, function of irf3. therefore, we demonstrate that compounds that activate ripa have potential for antiviral action. high throughput screening to isolate novel antiviral compounds is a widely accepted strategy, and commonly utilizes virus replication as a terminal readout. we took a novel approach to isolate the antiviral agents by screening for activators of a cellular antiviral pathway. irf3 is the key transcription factor for the induction of ifn and antiviral isgs [1, 4, 37, 38] . we have uncovered a pro-apoptotic pathway of nt-irf3, ripa, in virus-infected cells [7, 9, 13] . using cells and mice expressing a ripa active nt-irf3 mutant, we demonstrated the relative contribution of ripa to the antiviral response of irf3 [7] . several high throughput screens have revealed small molecules that regulate the transcriptional activity of irf3 [39] [40] [41] [42] [43] . however, because ripa is newly identified, there are no known regulators of this pathway. this led us to develop an unbiased screen, using a library of fda-approved drugs, to isolate ripa-activators that would function as antiviral agents. using doxorubicin, a new ripa activator, we examined both activities of irf3, because an activator of either pathway would provide an antiviral response. although doxorubicin promoted ripa in multiple cell types, it inhibited the transcriptional activity of irf3. therefore, doxorubicin inhibits virus replication in the absence of irf3-induced antiviral genes, further emphasizing the biological significance of ripa. how doxorubicin inhibits the transcriptional activity of irf3 is a topic of future investigation. the dna-damaging property of doxorubicin [44] may contribute to this activity. furthermore, whether doxorubicin, in addition to ripa promotion, can intercalate with viral genomic dna to inhibit viral replication, will require further studies. our screen isolated, in addition to doxorubicin, pyrvinium pamoate, which specifically promoted ripa, but not the transcriptional function, of irf3. many viruses block the transcriptional activity of irf3 to dampen ifn production [45] . in such scenarios, it will be interesting to examine whether ripa-promoting compounds inhibit virus replication. to investigate the molecular mechanisms of the doxorubicin-mediated promotion of ripa, we examined the key biochemical steps of the pathway. since viruses can block host pathways, we chose to use nonviral stimuli of rlr for the mechanistic studies, to avoid any virus-specific effects. our results indicate that doxorubicin did not enhance the irf3 ubiquitination nor the release of cytochrome c into the cytosol, the critical stages of ripa. interestingly, our results suggest that doxorubicin treatment may enrich a distinct pool of ubiquitinated irf3 in the rlr-stimulated cells. targeted proteomic analyses of this pool of irf3 will be done in the future to gain further insight. we also considered that doxorubicin, a dna-damaging agent, might stimulate ripa by triggering the sting signaling pathway via topoisomerase activation [46, 47] . the cgas/sting pathway can also trigger transcription-independent mitotic cell death by irf3 activation [48] . moreover, er stress-activated sting has been shown to trigger a ripa-like pathway in hepatocytes [17] . however, in our test cells, mda-mb-453, the sting agonists were unable to trigger apoptotic cell death, indicating a lack of sting contribution in promoting ripa. it remains to be seen whether the ripa activators also trigger additional innate signaling pathways, e.g., nlrs to amplify ripa activity. our results revealed a novel role of the erk signaling pathway in regulating ripa. sev infection activates cellular autophagy via the erk signaling pathway to trigger apoptosis [49] . whether doxorubicin or pyrvinium pamoate use a similar mechanism to promote ripa will require additional studies. our results further revealed that doxorubicin activated both erk and jnk signaling pathways in rlr-stimulated cells; however, jnk activity was not required for promoting ripa. in the future, in-depth genetic studies will reveal the relative contribution of erk and jnk to modulate ripa. the erk signaling pathway also regulates the transcriptional activity of irf3 [50] ; whether doxorubicin inhibits the transcriptional activity of irf3 via erk will require future investigation. in the future, in vivo studies in the context of virus-infection will be required to validate the in vitro findings regarding the erk signaling mechanism of ripa-activating drugs. a previous study indicated that p53, which can be activated by doxorubicin, is not involved in dsrna-induced cell death [31] . in previous studies, we have shown that sev-induced ripa is regulated by the pi3k/akt signaling pathway [15] . it remains to be seen, using nt-irf3 mutant that specifically activates ripa, whether doxorubicin also modulates the p53 and pi3k pathways to promote ripa. our screen, using a relatively small library of 1200 compounds, revealed that a number of molecules with diverse therapeutic activities promote ripa. in the future, we will expand the screen to larger libraries to isolate additional novel ripa-activating compounds. in addition, a screen using cells expressing nt-irf3 mutant that specifically activates ripa may reveal additional candidates. in the future, we will use mice expressing a ripa-active irf3 s1/s1 mutant to test the efficacy of the ripa-promoting drugs in mounting antiviral responses in the absence of antiviral genes. such studies will be physiologically significant by using the drug derivatives that are currently applied on patients. because doxorubicin inhibits the cellular ifn response, patients receiving this drug may be protected from the adverse effects of ifn and the ifn-induced genes. moreover, our study provides a molecular basis for the previously identified antiviral effects of the ripa-activating compounds [51, 52] . a recent study showed that pp is a novel inhibitor of coronavirus replication [53] and its antiviral property, in part, may be related to the ripa-promoting function of pp. whether activation of ripa directly inhibits virus replication or eliminates the virus-infected cells to reduce overall infection requires in-depth studies. some viruses trigger apoptosis for successful spreading and, therefore, promoting viral apoptosis to inhibit virus infection may be virus specific. finally, a strategy to repurpose ripa activators as antivirals must be met with caution, as there are a number of diseases where ripa can be detrimental (e.g., liver diseases). for instance, a ripa-like pathway in hepatocytes leads to the development of alcoholic liver diseases [17] . moreover, we recently showed that ripa-activated hepatic myeloid cells produce inflammatory cytokines that cause hepatitis in mice [18] . in these scenarios, it may be advantageous to investigate ripa inhibitors as therapeutic options. in summary, our results demonstrate that pharmacological activation of ripa may be a potential antiviral strategy, particularly in scenarios where the cellular ifn response is dampened. in the future, these ripa-modulating agents can be studied in additional models where ripa is involved, e.g., in fatty liver diseases. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/4/442/s1. figure s1 . representative data from the primary screen. a representative primary screening plate showing the caspase-3 activity in each well (rows and columns are shown in blue) containing a specific drug or dmso (vehicle) control. potential activator and inhibitor drugs are shown in the sample plate. nt, no treatment. figure s2 . validation of irf3−/− ht1080 cells. immunoblot of ifit3 protein expression in rlr-stimulated wt and irf3−/− (ko) ht1080 cells to ensure the gene knockout. figure table s1 . caspase activity of the primary screening plates. mda-mb-453 cells were transfected with polyi:c in the absence or the presence of the drugs, as described in figure 1f . caspase-3 activity of each well is indicated after normalizing to the vehicle (dmso) control, which was considered as 100. author contributions: conceptualization, s.c. and r.c.; methodology, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; software, s.c., a.g., and g.s.; validation, s.c. and a.g.; formal analysis, s.c., a.g.; investigation, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; resources, s.c., r.c. and r.t.t.; data curation, s.c., a.g., k.c., s.f., g.s., j.g., b.j., j.c. and r.t.t.; writing-original draft preparation, s.c., a.g. and k.c.; writing-review and editing, r.c., and r.t.t.; visualization, s.c., a.g., k.c., s.f. and g.s.; supervision, s.c., r.c. and r.t.t.; project administration, s.c., r.c. and r.t.t.; funding acquisition, s.c. and r.t.t. all authors have read and agreed to the published version of the manuscript. no love lost between viruses and interferons the irf family transcription factors at the interface of innate and adaptive immune responses regulating irfs in ifn driven disease convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response interferon-stimulated genes and their antiviral effector functions inhibition of viral pathogenesis and promotion of the septic shock response to bacterial infection by irf-3 are regulated by the acetylation and phosphorylation of its coactivators ubiquitination of the transcription factor irf-3 activates ripa, the apoptotic pathway that protects mice from viral pathogenesis role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by sendai virus viral apoptosis is induced by irf-3-mediated activation of bax irf-3 and bax: a deadly affair dsrna-activation of tlr3 and rlr signaling: gene induction-dependent and 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atg3-dependent manner disruption of erk-dependent type i interferon induction breaks the myxoma virus species barrier a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro inhibition of herpes simplex virus replication by anthracycline compounds high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank ganes sen, eain murphy, kevin pan, jyl matson and amit tiwari for help with critical reagents and resources used for the study. the authors would like to acknowledge funding from the national institutes of health grant aa026017 (sc), american heart association grant 15sdg25090212 (sc), medical research society (sc), and the university of toledo college of medicine and life sciences startup funds (sc and rtt). the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.viruses 2020, 12, 442 key: cord-262753-jld1ygxt authors: neidermyer, william j.; whelan, sean p. j. title: global analysis of polysome-associated mrna in vesicular stomatitis virus infected cells date: 2019-06-21 journal: plos pathog doi: 10.1371/journal.ppat.1007875 sha: doc_id: 262753 cord_uid: jld1ygxt infection of mammalian cells with vesicular stomatitis virus (vsv) results in the inhibition of cellular translation while viral translation proceeds efficiently. vsv rna synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mrnas that are structurally indistinct to cellular mrnas with respect to their 5′ cap-structure and 3′-polyadenylate tail. using the global approach of massively parallel sequencing of total cytoplasmic, monosomeand polysome-associated mrna, we interrogate the impact of vsv infection of hela cells on translation. analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. consistent with an overwhelming abundance of viral mrna in the polysome fraction, the reads mapping to cellular genes were reduced. the cellular mrnas that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more au rich, features that are shared with the viral mrnas. several of those mrnas encode proteins known to positively affect viral replication, and using chemical inhibition and sirna depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3a (eif3a)—encoded by 2 such mrnas—support viral replication. correspondingly, regulated in development and dna damage 1 (redd1) encoded by a host mrna with reduced polysome association inhibits viral infection. these data underscore the importance of viral mrna abundance in the shut-off of host translation in vsv infected cells and link the differential translatability of some cellular mrnas with proor antiviral function. introduction infection of mammalian cells by vesicular stomatitis virus (vsv) results in a profound shut-off of host cell gene expression. this host cell shut-off occurs at the level of mrna transcription through inhibition of rna polymerase ii by the viral-encoded matrix protein (m) [1] [2] [3] . the m protein also forms a complex with ribonucleic acid export 1 (rae1) and nucleoporin 98 (nup98) [4] thus suppressing host cell mrnp export from the nucleus, including that of mature cellular mrnas [5] [6] [7] [8] . vsv infection also inhibits protein synthesis by manipulation of the host-cell translation machinery, particularly at the level of translation initiation [9, 10] . eukaryotic initiation factor 4e (eif4e)-the rate limiting factor for translation initiation-recognizes the 7 m gpppn mrna cap structure as part of the eif4f complex, and in concert with other translation initiation factors facilitates the recruitment of the small 40s ribosomal subunit to the mrna prior to scanning to the initiating methionine where the 60s subunit joins [11, 12] . vsv infection results in the rapid dephosphorylation of eif4e itself, for which the functional consequences are unclear, and of its binding protein (eif4e-bp1) leading to eif4e sequestration and the suppression of translation initiation [9, 10] . viral gene expression evades the shut-off mechanisms employed to suppress host gene expression. as vsv rna synthesis occurs entirely within the cytoplasm, viral rna synthesis is not subject to the inhibitory effects of m on rna polymerase ii and mrna export from the nucleus. the vsv rna synthesis machinery comprises a ribonucleoprotein complex of the negative-sense genomic rna completely encased by a nucleocapsid protein (n) sheath and associated with the viral polymerase complex [13] . the viral transcriptase copies the n-rna template into 5 monocistronic mrnas that are structurally indistinct to those of the host-cell with respect to their 5 0 cap and 3 0 polyadenylate tail [14] [15] [16] [17] [18] [19] [20] . the enzymes necessary for mrna synthesis, namely an rna dependent rna polymerase (rdrp) and a set of capping enzymes, reside within the viral large protein (l) [17, 19, [21] [22] [23] [24] [25] [26] [27] . vsv l protein cannot engage the n-rna template directly, but instead depends on the viral phoshoprotein (p) to facilitate the interaction [28] [29] [30] [31] [32] [33] . messenger rna polyadenylation is also catalyzed by l through reiterative transcription by the rdrp of a u7 tract that resides at the end of each gene [34] [35] [36] [37] [38] . this program of viral transcription results in the cytoplasmic synthesis of 5 mrnas that depend upon the host machinery for their translation, and must therefore avoid the shut-down mechanisms that effectively suppress host mrna translation. metabolic labeling studies demonstrate that 4 hours post vsv infection of baby hamster kidney cells in culture, total translation is suppressed to about 65% the level of uninfected controls [39] . extraction of mrna from infected cells coupled with its in vitro translation confirmed that the cellular mrnas remain intact and are competent for translation [39] . the vsv mrnas are present in an approximately 2-3 fold excess of the total cellular mrna, leading to the model that competition between viral and cellular mrnas for ribosomes results in the dominance of viral translation [39, 40] . polysome analysis also demonstrates that the cellular mrnas are associated with significantly fewer ribosomes in infected cells [39] . for example, infection results in the movement of actin mrna from polysomes containing 12 or more ribosomes to those containing 5 [39] . this movement reflects the competition between viral and cellular mrna for ribosomes and the limited pool of eif4e. the competition model predicts that the kinetics of viral mrna synthesis and the levels of viral mrna should correlate closely with host shut-off. tests of this prediction yielded conflicting results. the kinetics of host shut-off and viral mrna accumulation correlate well for many strains of vsv, consistent with the competition model [39, 40] . inhibition of host protein synthesis is, however, largely unaffected following coinfection of cells with increasing quantities of defective interfering (di) particles that suppress viral mrna levels up to 14-fold [41] . a similar result was obtained for a vsv mutant that is restricted for genome replication at 39˚c, and yields only 30% of the wild type levels of viral mrna [41] . collectively, these studies suggest additional mechanisms may contribute to the shut-off of host cell protein synthesis. specific features of the viral mrnas that contribute to their efficient translation have not been defined. the 5 0 untranslated regions of vsv mrnas are short, being 10-15 nucleotides for the viral n, p and l mrnas that encode the proteins required for rna replication [42, 43] . how such short 5 0 utrs serve as effective initiators of translation is unclear. evidence for differential translation of viral mrna comes from small interfering rna suppression of eif4e, which inhibits host gene expression but has no impact on viral gene expression [44] . viral translation is also hypersensitive to the loss of ribosomal protein l40, suggesting different mrna features facilitate translation of viral versus host mrna [45] . flanking cellular or reporter genes by the conserved viral 10-nt gene-start and 13-nt gene-end sequences, and inserting them into the viral genome is sufficient to mediate their efficient translation [46] . by contrast, expression of the same genes following transfection of plasmid dna into cells and subsequent vsv infection does not offer this translational advantage [46] . thus, transcription of the mrnas from the viral genome appears to contribute to their efficient translation. in the present study, we interrogate global mrna translation in vsv infected cells using rnaseq analysis of the cytoplasmic mrna transcriptome, and parallel sequencing of polysome-associated mrnas. we obtain support for the model that an overabundance of viral mrna contributes to host shut-off by leading to a re-distribution of cellular ribosomes onto viral mrna. by combining this rnaseq analysis with examining the distribution of specific viral and cellular mrnas within polysomes, we also demonstrate that mrnas shift to smaller polysomes. analysis of cellular mrnas less-sensitive to this global shut-down of translation identifies several host proteins that promote viral replication. similar analyses revealed the abundance of viral mrna contributes to the host-cell shut-off for other viruses including coronaviruses, influenza and vaccinia [47] [48] [49] . to interrogate the impact of vsv infection on global translation we isolated total cytoplasmic, monosome-and polysome-associated mrna from hela cells at 2 and 6 hpi and compared the relative sequence reads obtained by deep-sequencing ( fig 1a) . statistical analysis of sequencing reads between biological replicates from each fraction yields a pearson correlation of >0.97 for cytoplasmic, monosome-and polysome-associated mrna pools validating reproducibility between the replicates. as visible in the polysome profiles (fig 1b) , vsv infection results in a small but reproducible increase in the pool of monosomes and large polysomes at 2 hpi, and a collapse of large polysomes and an increase in monosomes by 6 hpi. mapping of the sequence reads to the viral and host genome highlights that by 6 hpi >60% of the total reads in the cytoplasmic and polysome fractions are viral (fig 1c) . this increase from the <1% observed at 2 hpi (fig 1c) emphasizes the impact of the exponential phase of viral rna replication and secondary transcription of the viral genome on mrna production. the viral sequence reads map to all 5 genes, with clear dips in coverage at gene-junctions (s1 fig) . consistent with the order of transcription of the viral genome and the localized transcriptional attenuation at gene-junctions [50] [51] [52] [53] , the relative reads that map to each viral gene generally diminish with distance from the single 3 0 promoter ( fig 1d and s1 fig) . analysis of the sequencing reads that map to cellular genes supports that like the viral mrnas, the level of reads in the polysome fraction mirrors that in the total cytoplasmic fraction at 2 and 6 hpi ( fig 1c) . this result demonstrates that the majority of mrnas are polysome-associated in proportion to their abundance. the abundance of the 5 viral mrnas at 6 hpi supports the model that viral mrnas outcompete cellular mrnas for ribosomes [39] . we note that viral mrnas are, however, underrepresented (49%) and cellular mrnas overrepresented (51%) in the monosome fraction at 6 hpi, compared to their cytoplasmic abundance ( fig 1c) . this finding is consistent with a differential effect on viral versus host mrna translation. to determine how vsv infection affects the distribution of the population of mrnas between total cytoplasmic, monosome or polysome fractions, we plotted the transcript per million (tpm) for each individual mrna mapped to the human and viral genome in all 3 fractions (fig 2) . at 2 hpi, reads that map to the viral genes in each fraction are similar in abundance to those reads that map to highly expressed cellular genes (fig 2a) . the reads that map to any given cellular gene alter within a relatively narrow range, with few genes showing a greater than 2-fold change in the relative number of sequence reads (fig 2b) . for the population of mrnas, the relative reads obtained from the polysome fraction mirrored the relative reads in the total cytoplasmic fraction, consistent with the abundance of an mrna being a determinant of its translatability. by 6 hpi, reads that map to each of the 5 viral genes-with the exception of l-exceed the reads that map to any individual cellular gene (fig 2c, red triangles) . this is concurrent with a decrease in reads that map to the majority of cellular genes in cytoplasmic, monosome and polysome-associated fractions (fig 2c) . there were, however, some distinctions between the monosome and polysome fractions. for the majority of the population of cellular mrnas, hela cells were infected with vsv at a moi of 10 and cytoplasmic extracts were prepared at 2 and 6 hpi for mrna isolation and polysome profiling. messenger rna was isolated from fractions corresponding to 80s monosomes, or polysomes containing 3 or more ribosomes, and used for deep sequencing. (b) polysome analysis of uninfected (black) or vsv (red) infected hela cells. cytoplasmic extracts were sedimented through a 10-50% sucrose gradient and 0.5 ml fractions were collected while continuously monitoring absorbance at λ = 254nm. (c) distribution of fragments mapping to the concatenated hg38 (human) and vsv genomes for cytoplasmic, monosome, and polysome samples at 2 and 6 hpi. trimming and mapping was performed in clc genomics workbench. (d) distribution of reads among the 5 viral genes at 2 and 6 hpi. expression level is presented as transcripts per kilobase million (tpm) to normalize for gene length and library size, error bars denote standard deviation from two biological replicates. reads were most reduced in the polysome fraction compared to the total cytoplasmic fraction ( fig 2d) . a smaller reduction in reads was observed in the monosome fraction, and some cellular mrnas even showed an increase in reads compared to the total cytoplasmic fraction ( fig 2c) . this may reflect differences in the movement of cellular mrnas from large polysomes to monosomes or out of the pool of translating ribosomes. we next mined our sequence data for evidence of differential translation of cellular mrnas following vsv infection. for this analysis we divided the polysome tpm by the total cytoplasmic tpm as an indicator of the efficiency with which any given mrna is translated. we also performed a similar analysis for the monosome pool. we are cognizant of the fact that such ratios ignore the movement of any given mrna from larger to smaller polysomes, and will likely represent an underestimate of the extent of any translational regulation. to identify the subset of the population of cellular mrnas with the highest probability for translational regulation in infected cells, we plotted the fold change in tpm at 2 and 6 hpi (fig 3a-3d ). at 2 hpi the monosome or polysome-associated reads changed within a narrow range for the majority of cellular genes ( fig 3a) . the marked shut-off of host protein synthesis observed by metabolic labeling suggests that at 6 hpi the association of cellular mrna with polyribosomes would alter significantly at the population level. although we observe a global reduction in polysome-associated reads for the bulk of the population of cellular mrnas the reduction is less than 2-3 fold. accompanying this global reduction in polysome-associated reads, we also observe an increase in monosome-associated reads with more than half the mrnas within the population exhibiting a >2-fold increase (fig 3b and 3d ). from the above ratios we selected the subset of cellular mrnas that exhibit the largest changes in relative polysome-associated reads at 6 hpi to determine whether those mrnas shared any common features. for this purpose, we selected those mrnas that change >2 standard deviations of the mean and thus exceed the 95% confidence interval. this analysis identified 364 cellular mrnas as candidates for translational upregulation and 138 cellular mrnas as candidates for translational downregulation following vsv infection (fig 3b and 3d ). consistent with monosome and polysomeassociated reads at 2 hpi changing within a narrow range, only 4 genes with increased and 20 with decreased polysome association, overlap between 2 and 6 hpi (fig 3c and 3d ). within the monosome fraction 8 genes with increased and 6 with decreased monosome association overlap from 2 to 6 hpi. we next determined whether shared functional or sequence elements are present within the specific subsets of 364 mrnas with increased polysome association, or the 138 mrnas with decreased polysome association (fig 3b and 3d , blue and green dots). for the 364 genes with significantly increased polysome-associated reads, gene ontology analysis identifies functions in rna binding, helicase and ntpase activities, among others ( fig 3e and s3 dataset) . the 138 genes with decreased polysome-associated reads are associated with cellular responses to stimuli and signaling activities ( fig 3f and s3 dataset) . this gene ontology analysis reveals that the up and down regulated transcripts comprise distinct functional groups. at 6 hpi the cytoplasmic abundance of cellular mrnas correlates with their polysome association at 6 hpi ( fig 4a and 4b ), consistent with mrna abundance being a determinant of translatability. as described above, we use as an indicator of translation efficiency (te) of an mrna the ratio of polysome to total cytoplasmic associated reads. to determine whether there are shared features between the 364 mrnas with evidence of enhanced polysome association or the 138 with reduced polysome association, we extracted mrna sequences and annotations from the ucsc genome browser. assisted by published datasets we examined whether the half-life, size, gc content or poly(a) tail length correlate with increased or decreased polysome association (fig 4c-4f and s2 fig) [54, 55] . cellular mrnas with increased polysome-associated reads tended to have a longer half-life, were typically larger, and were more au-rich ( fig 4c-4e and s2 fig) . correspondingly, those with decreased polysome-associated reads tended to have shorter half-lives, higher gc content, and were typically smaller. the correlation between higher au content and increased polysome-associated reads was most evident for the coding region and 3 0 utr (s2 fig). the effect of length was predominantly a determinant of the orf and not the 5 0 or 3 0 utr (s2 fig). there was no correlation between poly(a) tail length and polysome-associated reads at 6 hpi ( fig 4f) . this analysis highlights that the cellular mrnas that exceed the 95% confidence interval for increased polysome-associated reads in response to vsv infection are most similar to the viral mrnas in that they are typically longer and more au rich. we next examined whether cellular mrnas that exhibit increased polysome association encode proteins that are pro-or antiviral. among the 364 cellular mrnas with increased polysome association several encode known proviral factors including the heat shock proteins (hsp) 90, 70, and 60. previous work demonstrated that inhibition of hsp90 inhibits viral replication, and linked inhibition of those chaperones to defects in l protein folding [56] [57] [58] . we independently verified the proviral function of hsp90 using the inhibitor 17-[2-(dimethylamino)ethyl]amino-17-desmethoxygeldanamycin (17-dmag) [59, 60] . infection of hela cells with vsv that expresses egfp as a marker of infection demonstrates that 17-dmag has no effect on the fraction of cells infected, but slows the rate of egfp expression ( fig 5a and s3 fig) . this was not simply due to defects in egfp folding, as metabolic labeling of viral rna substantiates the defect in gene expression (s3 fig) . we also found that polysome association of the mrna encoding eukaryotic initiation factor 3 subunit a (eif3a), increases after infection. to test whether this reflects a specific proviral function of eif3a, we used sirna depletion to reduce eif3a and measured viral gene expression using reporter viruses expressing egfp or luciferase. both reporter viruses displayed a sensitivity to the loss of eif3a (figs 5b and s3). as expected, depletion of eif3a also reduced cellular translation in uninfected cells, but that reduction was modest as evidenced by expression of a cmv promoter driven renilla luciferase reporter ( fig 5b) . translation of the cmv driven reporter, however, reflects the accumulated steady-state levels of luciferase mrna. we therefore measured the effect of eif3a depletion on viral vs host translation by metabolic incorporation of [ 35 s]-methionine in infected and uninfected cells (fig 5b) . following eif3a depletion we observed a 55% reduction in viral m protein synthesis over a 30 minute time period, which is similar to the 56% reduction in host protein synthesis measured in mock infected cells. this analysis supports a proviral role for 2 cellular mrnas that encode proteins with important house-keeping functions that remain polysome-associated following vsv infection. among the 138 cellular mrnas that exhibit reduced polysome association following infection was dna-damage inducible transcript 4 (ddit4) which encodes regulated in development and dna-damage response 1 (redd1) [61] [62] [63] . existing studies demonstrate that ddit4/redd1 restricts the replication of negative-strand rna viruses, including vsv. depletion of ddit4/redd1 by sirna increased viral gene expression as evidenced from infection of cells with vsv-egfp, and by metabolic labeling of viral protein synthesis (fig 5c, s3 fig) . consistent with the enhancement of viral gene expression following ddit4 depletion, we obtained an approximately 10-fold increase in viral titers ( fig 5c) . depletion of ddit4/redd1 also increases cellular protein synthesis likely reflecting its role as a negative regulator of mtor ( fig 5c and s3 fig) . the above analysis supports that the polysome association of some host mrnas following vsv infection correlates with their pro-or antiviral functions, but does not directly demonstrate that the level of polysome association is associated with a change in synthesis of the corresponding protein. to independently examine whether changes in polysome association of host mrnas affect synthesis of the corresponding protein we selected the heat shock protein (hsp70) and y-box binding protein 1 (ybx1) as representative mrnas with increased and decreased polysome association, respectively. we selected those mrnas based on their highlevels of expression, stability, and availability of antibodies suitable for the selective immunoprecipitation of the corresponding protein. we compared the effect of vsv infection on protein synthesis by selective immunoprecipitation of proteins following metabolic incorporation of [ 35 s]-methionine from 3-6 hours post infection (fig 5d) . synthesis of hsp70 3-6 hpi is indistinguishable to that synthesized during a 3h period from mock infected cells (fig 5d) . by contrast, ybx1 synthesis decreases more than two-fold ( fig 5d) . this result confirms for 2 cellular mrnas that the extent of polysome association observed by our rnaseq analysis is reflected in synthesis of the corresponding host proteins. analysis of cellular mrnas with high cytoplasmic abundance (purple) or low cytoplasmic abundance (orange) as compared to mrnas with cytoplasmic abundance within 2 standard deviations of the mean abundance (gray) in uninfected cells. cytoplasmic abundance by tpm is from the data set published with this paper. ��� p< 2.2 x 10 −16 ; �� p< 5.0 x 10 −5 ; � p< 0.05; all others p> 0.05 as determined by the wilcoxon rank sum test compared to mrnas with relative abundance levels within the 95% confidence interval of the mean. hinges correspond to the 25th-75th percentiles, and whiskers denote 1.5 times the inter-quartile range. (b) analysis as in a for cytoplasmic abundance in infected cells. (c-f) mrna characteristics for mrnas with increased polysome association (blue) or decreased polysome association (green) at 6 hpi, as defined in fig 3. data for rna half-life and poly(a) tail length were from [54, 55] . analysis was performed as in a. for our experiments we pooled fractions that contained 3 or more ribosomes prior to sequencing of the polysome-associated mrna. as a result, we do not assess the impact of the redistribution of mrnas toward smaller polysomes. we therefore selected a subset of cellular mrnas (fig 6a) , and interrogated their distributions across polysomes using reverse transcription and quantitative pcr. as controls, we analyzed the distribution of n and g mrnas as representative viral transcripts translated by soluble and endoplasmic reticulum-associated ribosomes, respectively [64] . consistent with the robust production of viral proteins at 6 hpi, the vsv n and g mrnas were localized in fractions corresponding to 3 or more ribosomes (fig 6b) . for two cellular mrnas with increased polysome tpm-collagen type iv alpha 2 (col4a2) and alpha-actinin-4 mrna (actn4)-the mrnas remained associated with larger polysomes in infected cells (fig 6c) . two cellular transcripts that were largely unaltered in their polysome associated tpm-β-actin (actb) and glyceraldehyde 3-phosphate dehydrogenase (gapdh)-remained polysome-associated, although there was a shift toward smaller polysomes and some gapdh transcripts exited polysomes (fig 6d) . for two representative cellular mrnas with decreased polysome tpm-transforming growth factor b induced factors (tgif1) and ubiquitin conjugating enzyme e2 b (ube2b)-the mrnas largely exited the polysome fractions, and those that remained were predominantly present on smaller polysomes ( fig 6e) . in all cases examined, dissociation of polysomes with edta shifted the mrna distribution toward the fractions corresponding to free ribosomal subunits (s4 fig). these qpcr data highlight the shift towards smaller polysome fractions for some cellular mrnas, which also likely contributes to suppression of host protein synthesis. this shift might also explain our finding that hsp70 protein synthesis is relatively unaffected by viral infection (fig 5d) , although the mrna exhibits increased polysome association. the abundance of viral mrna and the suppression of translation initiation through reducing the pool of eif4e will both contribute to the movement of mrnas toward smaller polysomes. recognition of the mrna cap-structure by eif4e requires that the guanine-n-7 position of the 7mgpppnmn cap is methylated [65] . we previously reported a panel of recombinant vsvs containing amino acid substitutions within the l-encoded mrna cap methylase domain that are defective in viral mrna cap methylation [25] . mutants vsv-l g4a and vsv-l g1670a contain substitutions in the binding site for the methyl donor s-adenosyl methionine (sam) and ablate all viral mrna cap methylation (vsv-l g4a ) or guanine-n7 but not ribose-2 0 -o methylation (vsv-l g1670a ) [25] . as vsv mrna is relatively insensitive to the loss of eif4e [44] , we would anticipate that the methylation status of the mrna cap-structure would have little impact on polysome association. analysis of the distribution of vsv n and g mrna within polysomes at 6 hpi revealed a similar distribution in cells infected with wild-cells infected with wild-type vsv. the position of viral proteins is noted to the right. presented is a representative gel from two independent replicates. (c) vsv gene expression and replication in ddit4 depleted cells. a representative histogram of egfp expression is shown along with the mfi of cells normalized to a non-targeting sirna control. error bars denote the standard deviation from the mean of three independent replicates. for metabolic labeling a representative gel of two independent replicates is presented. kinetics of viral replication were measured by titration of yields at various times post infection of sirna treated hela cells. error bars denote the standard deviation from the mean of 3 independent replicates. (d) immunoprecipitation of cellular proteins synthesized post-infection with vsv wt . shown is a representative gel from two independent replicates. a quantitative analysis of the hsp70 and ybx1 bands is shown in the bottom two panels, error bars denote the standard deviation from two independent replicates. https://doi.org/10.1371/journal.ppat.1007875.g005 control of vsv protein synthesis type virus as well as those infected with vsv-l g1670a and vsv-l g4a (fig 7a-7c) . correspondingly, the rate of viral protein synthesis in cells infected with vsv-l wt and vsv-l g1607a measured by a 10-minute pulse of [ 35 s]-methionine is similar (s5 fig). these results demonstrate that defects in viral mrna cap methylation do not significantly alter the rate of viral protein synthesis, consistent with a reduced dependence on eif4e [25] . to directly test whether manipulating eif4e levels affects viral translation we depleted eif4e levels approximately 10-fold using a peptide-conjugated morpholino (ppmo) and measured the rates of vsv-l wt and vsv-l g1607a viral protein synthesis by a 10-minute pulse of [ 35 s]-methionine at various times post-infection (fig 7d-7f) . depletion of eif4e decreased the rate of viral protein synthesis in vsv-l 1670a infected cells, but not l wt infected cells ( fig 7e and 7f) . this was not due to sequestration of eif4e by differential activation of eif4e-bp1 between vsv-l wt and vsv-l g1607a infected cells, as the kinetics of eif4e-bp1 dephosphorylation are the same during both infections (s6 fig). we previously reported that although vsv-l 1670a is defective in mrna cap methylation, up to 20% of the mrna cap-structures are guanine-n7 methylated. we interpret this finding as indicative of an eif4e dependent mechanism of translation early in infection. we obtained two snapshots into the complex battle for control of protein synthesis in cells infected with vesicular stomatitis virus by sequencing of polysome-associated mrnas at 2 and 6 hpi. those snapshots provide further evidence that the abundance of vesicular stomatitis virus mrnas is a determinant of the dominance of viral protein synthesis in infected cells, but highlight several additional attributes of this complex relationship. those include the demonstration that some host mrnas that remain polysome associated encode proteins that support viral replication, and some of those that exhibit reduced polysome association encode proteins that are antiviral. we also obtained further insight into the seemingly paradoxical observations that viral infection results in a reduction of the available pool of eif4e -the rate limiting factor for translation initiation-yet viral mrnas contain a cap structure that is indistinct to that of host mrnas. through the use of a viral mutant defective in mrna cap methylation, and suppression of eif4e levels we provide evidence consistent with a transition from an eif4e dependent phase of viral translation to one less-dependent on eif4e. the sequence data reported here provides some support for the model that the vsv mrnas overwhelm the pool of cellular mrna leading to a redistribution of ribosomes onto viral messages [39] . evidence in support of this model is based on massively parallel sequencing of mrnas associated with polysomes, compared with those present in the cytoplasm. as a fraction of the total cytoplasmic mrna, the vsv mrnas represent~1% by 2 hpi, but more than 60% by 6 hpi, illustrating the power of exponential amplification of the viral genome. as a result, the viral n, p, m and g mrnas far exceed the abundance of any given cellular mrna, and even the least abundant viral mrna-that encoding the l polymerase-is present at similar levels to the most abundant cellular mrna. thus, one contributor to host cell shut-off in vsv infected cells appears to relate to the synthetic capabilities of the viral polymerase in transcription of viral mrna. similar conclusions have recently been reached for other viruses. infection of cells with mouse hepatitis virus (mhv) a positive-strand rna coronavirus that replicates within the cytoplasm results in 80-90% of the cytoplasmic mrna being viral by 5 hpi [47] . for influenza a virus, a segmented negative-strand rna virus that replicates in the nucleus, >50% of the total mrna in the cytoplasm is viral [48] . in this case however, the viral endonuclease pa-x degrades cellular mrna which further contributes to the dominance of viral mrna [66, 67] . for vaccinia virus, a dna virus that replicates entirely within the cytoplasm, degradation of host mrna through the viral encoded decapping enzymes d9 and d10 also helps the viral mrnas overwhelm those of the host cell [49, 68, 69] . collectively these studies indicate that one shared mechanism for host cell shut-off in virus-infected cells is competition for host cell ribosomes through tipping the balance between viral and host mrna. earlier work concludes that viral mrna abundance is not the determinant of host cell shut-off [41] . when vsv mrna levels were suppressed up to 14-fold by using defective interfering particles of vsv or a viral mutant defective in transcription, host shut-off was still observed. we did not directly test how suppressing viral mrna levels impacts host shut-off in this study, but rather conclude that abundance is only part of the mechanism by which the virus induces host cell shut-off-as discussed below. we also obtained evidence in support of additional mechanisms that contribute to host cell shut-off in vsv infected cells. we confirmed earlier work that demonstrated a suppression of the pool of the rate limiting factor for initiation, eif4e, by altering the phosphorylation status of its negative regulator, eif4e-bp1, which results in eif4e sequestration [9] . differences in sensitivity to reductions in eif4e may contribute to the overrepresentation of cellular mrna we observe in monosome fractions during infection. we also provide new evidence in support of a phase of vsv gene expression that is dependent on eif4e through the use of a viral mutant partially defective in guanine-n7 methylation [25] and by the suppression of cellular pools of eif4e. when eif4e levels are suppressed 10-fold, we unmask a defect in viral protein synthesis in cells infected with vsv-vsv-l g1670a a mutant with a 4-fold defect in methylation at the guanine-n7-position of the cap-structure. we suspect that this significantly underestimates the eif4e dependent phase of viral replication since transformed cell lines, like the hela cells used here, have higher constitutive levels of eif4e [70] . our findings are consistent with a model where viral mrnas initially compete with cellular mrnas and translate in an eif4e dependent manner. as infection progresses and the shut-down of host transcription, mrna export and eif4e sequestration continue, the process of initiation is increasingly less dependent on eif4e. the mechanism by which the viral mrnas become less dependent on eif4e remains uncertain, but earlier studies demonstrate that neither the 5 0 or 3 0 utr of viral mrnas facilitate this efficient translation. ongoing transcription of viral mrna from the viral genome has also been linked to efficient protein synthesis [46, 71] . whether this reflects the fact that the virus is an efficient producer of mrna that supports the competition model, or whether there is a temporal requirement for continued viral mrna synthesis is uncertain. as obligate intracellular parasites, viruses depend upon host cell functions for their replication. our sequence analysis provides evidence that vsv infection differentially impacts the polysome association of cellular mrnas. several host mrnas increased in polysome association include genes with known "proviral" functions for entry and replication including heparan sulfate, clathrin, and hsp90 [56, [72] [73] [74] . similarly, host mrnas with decreased polysome association included genes with published roles in restricting vsv replication such as interferon regulatory factor 1 (irf1), ddit4, and txnip [62, 63, 75] . it is difficult to definitively determine whether this reflects evolution of the virus to contend with the environment in which it finds itself or a bona-fide pro and antiviral effect of a given host protein. our efforts to address this are confounded by the essential house-keeping nature of many of the proteins encoded by host mrnas that remain polysome associated. an example of this is provided by enhanced polysome association of eif3a on vsv infection-a protein that is required for assembly of the multisubunit eif3 complex. that complex also includes eif3d which has demonstrated cap-binding ability, and directs eif4e-independent translation of select mrnas [76] [77] [78] [79] . depletion of eif3a, however, resulted in an equivalent reduction in the rates of viral and host translation-inconsistent with a specialized need for eif3 components in vsv protein synthesis. in this study, we validated that the effect of vsv infection on the polysome association of 2 host mrnas-hsp70 and ybx1-had a concordant impact on protein synthesis. although synthesis of hsp70 did not increase per se, this is likely explained by the shifting of many cellular mrnas towards smaller polysomes. this finding highlights the fact that the designation of rnas as having "increased" or "decreased" polysome association is imprecise, and reflects the complexities of how any given host gene is regulated. nevertheless, the general finding that mrnas with "increased" polysome association on vsv infection are typically larger, have longer half-lives and higher au content-features that are shared with the viral mrna-highlights commonalities among mrnas that remain polysome-associated and thus are more efficient in competing for ribosomes during host shut-off. the cellular mrnas that exhibit reduced translation efficiency were shorter, have shorter half-lives and higher gc content. although we validated changes in translatability and differential impacts on viral gene expression for a few cellular genes, it would be of significant interest to perform stable isotope labelling by amino acids in cell culture (silac) to non-radioactively label newly synthesized cellular proteins, quantify them on a genome-wide scale and correlate those data with the rnaseq results presented here. in addition to suppression of host translation through mrna synthesis and eif4e manipulation, the vsv matrix protein inhibits host rna polymerase ii transcription [1, 3] , and blocks nuclear export of mature mrnas through complex formation with the nuclear pore components rae1 and nup98 [4] [5] [6] [7] [8] . a well characterized viral mutant (vsv-m m51r ) fails to interact with the nuclear pore complex and exhibits a delayed kinetics of host shut-off [6, 10, 80] . a similar analysis to that described here of cells infected with such a mutant may help delineate the extent to which ongoing synthesis and export of cellular mrna impacts host cell shut-off. we anticipate that over the time course of vsv infection, the contribution of ongoing synthesis and export of host mrna from the nucleus will result in a relatively modest increase in the fraction of the cytoplasmic mrna that is cellular. this study highlights a strategy shared among distinct viruses to commandeer the host translational machinery by outcompeting cellular mrnas. precisely where the tipping point between viral and host mrna levels with respect to this shut-off occurs is uncertain. for vsv, a viral mutant that makes less mrna than the wild type yet still exhibits host cell shut-off suggests that shut-off can be achieved with less than the 60% of total cytoplasmic mrna observed here [41] . additional work will be required to define whether a specific tipping point exists and how this is influenced by other viral strategies such as eif4e suppression or blocking host gene transcription. hela cells (a gift from james hogle) were maintained in dulbecco's modified eagle medium (dmem; invitrogen, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs; tissue culture biologicals, tulare, ca). viral stocks were grown on syrian golden hamster kidney bsrt7 cells (a gift from k. conzelmann), and purified on linear 15-45% sucrose gradients prepared in nte (10 mm tris ph 7.4, 100 mm nacl, 1 mm edta). viral titers were determined by plaque assay on african green monkey kidney vero cells (atcc, ccl-81). for infections, cells were first washed with hanks' balanced salt solution (hbss) and incubated with virus for 1 hour at 37˚c in serum free medium, washed with hbss and subsequently incubated with medium supplemented with 2% fbs. for polysome profiling, hela cells were treated with dmem containing 100 μg ml -1 cycloheximide at 37˚c for 3 minutes. cells were washed twice with 1x ice-cold phosphate buffered saline (pbs) containing 100 μg ml -1 cycloheximide, and kept on ice or at 4˚c. cells were scraped into a 1.5 ml microcentrifuge tube in 1x pbs with 100 μg ml -1 cycloheximide, and pelleted at 300 × g for 10 minutes. cells were resuspended in 250 μl of a hypotonic buffer of 5 mm tris (ph 7.4), 2.5 mm mgcl 2 , 1.5 mm kcl, and rnasin (promega, madison, wi), supplemented with cycloheximide to 100 μg ml -1 and dl-dithiothreitol (dtt) to 3 μm. the detergents triton x-100 0.5% (vol/vol) and sodium deoxycholate 0.5% (wt/vol) were then added sequentially, cells were briefly vortexed, and incubated for 15 minutes on ice and clarified by centrifugation at 12,000 × g for 2 min. polysomes were separated on sucrose gradients prepared on a gradient master station (biocomp, fredericton, canada) using 10% and 50% (wt/ vol) sucrose dissolved in 15 mm tris (ph 7.4), 15 mm mgcl 2 , and 150 mm nacl supplemented with rnasin and 100 μg ml -1 cycloheximide. following centrifugation for 2 hours at 40,000 × g in a beckman coulter ultracentrifuge using an sw40ti rotor, 500 μl fractions were collected from the top of the gradient while monitoring absorbance at λ = 254 nm on a gradient master station. rna was extracted from total cytoplasmic, polysome, or monosome fractions using ls trizol (invitrogen) according to the manufacturer's protocol. equal amounts of rna as determined by spectrophotometry using absorbance at 260 nm on a nanodrop 2000 (thermo fisher, waltham, ma) were subject to library preparation using the illumina truseq vii rna library preparation kit (illumina, san diego, ca), and sequenced at the whitehead institute (cambridge, ma) on an illumina hiseq2500. reads were trimmed and mapped to the concatenated hg38 and vsv genomes using clc genomics workbench (qiagen, redwood city, ca). mapping parameters were as follows; mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.8, similarity fraction 0.8, and a maximum of 10 hits per read. raw sequence data is available from the ncbi sequence read archive (sra) under the primary accession code srp158625. transcripts per kilobase million (tpm) was calculated for genes with 56 or more mapped reads in the cytoplasmic fraction of both uninfected and infected cells using the total number of mapped exon reads. to identify cellular mrnas that were potential targets for translational regulation in infected cells, we determined the tpm in the polysome fraction/tpm in the total cytoplasmic fraction for each individual mrna. this ratio was determined for uninfected and infected cells, and presented as the log 2 fold change. gene ontology analysis was performed in r using goseq. utrs and cds sequences were downloaded from the ucsc table browser using "knowncanonical" mrna identifiers. non-protein coding rnas were excluded from the analysis. poly (a) tail length and mrna half-lives were from published data sets [54, 55] . graphs and statistical analyses were performed in r using the "wilcox_test" statistical test, the "density" kernel density estimation, and "geom_boxplot" or "geom_density" functions in ggplot and cowplot. total rna was recovered from polysome fractions using ls trizol according to the manufacturer's protocol. rna (500 ng) was annealed with oligo d(t) 20 and reverse-transcribed using superscript iii reverse transcriptase (thermo fisher) at 50˚c for 1 hour. following digestion of the rna strand with rnasea and rnaseh for 15 min at 37˚c, reactions were diluted 1:5 for cellular gene-specific qpcr or 1:125 for viral gene-specific qpcr. quantitative pcr was performed using power sybr green (thermo fisher) and relative amounts determined by images of rvsv-egfp infected cells were acquired using a 10× objective on a nikon eclipse te300 microscope (nikon instruments, melville, ny) equipped with a spot rt se18 monochrome camera (diagnostic instruments, sterling heights, mi). for cytometry, cells were washed in hbss, trypsinized, fixed in 4% paraformaldehyde at 4˚c for 15 minutes and measured using a facscalibur (cytek development, freemont, ca). cytometry data was analyzed using flowjo (flowjo inc, ashland, or). for mean fluorescence intensity (mfi) we gated on live cells identified by forward and side-scatter. to measure the % infected cells we subtracted those cells that fell within the gate established from uninfected control cells. for luciferase assays, where indicated hela cells were transfected with sirna, and the medium replenished at 24 h. cells were transfected 6 h later with 25 ng prl-cmv (promega), and activity measured 24 h later. for viral driven luciferase reporters sirna transfected cells were infected at 48 h and monitored 6 h later. luciferase expression was measured in a spec-tramax l microplate reader using the appropriate reagents according to the manufacturer's instructions (promega, e1501 and e2810). for depletion of host factors by peptide-conjugated morpholinos (ppmos) approximately 2.0 x 10 4 hela cells per well of a 24 well plate were treated 24 h later with 15 μm of the indicated ppmo. at 24 h post treatment, the media was replaced with fresh medium containing 15 μm ppmo, and used for testing 24 h later. cells were washed twice with ice-cold 1x pbs and lysed in rose lysis buffer consisting of 10 mm tris-hcl (ph 7.4), 66 mm edta, 0.4% w/v sodium deoxycholate, and 1% v/v np-40 on ice for 15 minutes. rose lysis buffer was supplemented with phosphatase inhibitor cocktail 2 (sigma-aldrich, st. louis, mo) and halt protease and phosphatase inhibitor cocktail (thermo fisher) for detection of phospho-eif4e-bp1. lysates were clarified, protein input was normalized by bradford assay and proteins resolved on polyacrylamide gels-10% for eif3a and eif4e or 12%, eif4e-bp1. proteins were transferred to nitrocellulose membranes for 90 minutes, eif4e and eif4e-bp1, or 120 minutes, eif3a, at 100v. membranes were blocked with odyssey blocking buffer in pbs (li-cor, lincoln, ne) for 1 h at room temperature, and incubated overnight at 4˚c with the following primary antibodies: rabbit anti-eif3a (cell signaling, #3411), rabbit anti-eif4e (cell signaling, #9742), rabbit anti-4e-bp1 (cell signaling, #9452), rabbit anti-phospho-4e-bp1 ser65 (cell signaling, #9451), rabbit anti-phospho-4e-bp1 thr37/46 (cell signaling, #2855), mouse anti-actin (millipore, #mab1501), mouse anti-actin (sigma, #a5316). membranes were washed 3x with 1x pbs-t for 5 minutes at room temperature, and incubated with the relevant secondary antibodies: goat anti-mouse irdye 680rd (li-cor, #925-68070) or goat anti-rabbit irdye 800cw (li-cor, #925-32211), for 1 hour at room temperature. membranes were washed again and kept in 1x pbs, and scanned on an odyssey clx (li-cor). hela cells were starved in dmem lacking l-methionine (corning, #17-204-cl) for 30 minutes, prior to addition of [ 35 s] express protein labeling mix (perkin elmer, waltham, ma) at 0.11 mci ml -1 . cell lysates were prepared as described above and separated on a low-bis 10% polyacrylamide gel. the gel was dried for 1.5 h at 80˚c in a vacuum gel drier and detected using a phosphoimager. quantitative analyses of band intensities was performed in image-quant tl v8.1 (ge healthcare, marlborough, ma). for radioimmunoprecipitations, 4.0 x 10 6 hela cells plated in 10 cm dishes (corning) were starved of methionine for 1 h at 24 h post-plating, and labeled with [ 35 s]-express for 3 h. cells were washed twice with ice-cold 1x pbs, collected by scraping and subsequent centrifugation for 2 minutes at 4˚c and 2,000 × g and lysed in 1 ml of 50 mm tris (ph 7.4), 150 mm nacl 2 , 1 mm edta, 1% v/v np40, 2 mm dtt, supplemented with pierce protease inhibitor (thermo fisher) on ice for 15 minutes. protein input was normalized by bradford assay, sds was added to 0.1%, and 450 μl lysate was pre-cleared for 1h at 4˚c on a nutator with 50 μl prewashed pierce protein a agarose beads. protein a beads were pelleted by centrifugation for 2 minutes at 4˚c and 2,000 × g and the labeled supernatant incubated with primary antibody at 4˚c overnight. the antibodies used for immunoprecipitation were 4 μg anti-yb1 (abcam, #ab76149) and 5 μg anti-hsp70 (enzo, #adi-spa-822). immune complexes were isolated using 50 μl pre-washed pierce protein a agarose beads, by incubating for 4 hours at 4˚c. bead complexes were collected by centrifugation for 2 minutes at 4˚c and 2,000 × g, washed 5x with 950 μl ice-cold ip lysis buffer with 0.1% sds, on an orbital shaker for 3 minutes at room temperature. protein-antibody complexes were eluted by boiling in 4x sds loading buffer, the beads pelleted for 2 minutes at 4˚c in a microcentrifuge and the supernatant loaded on a 10% polyacrylamide gel. after electrophoresis the gel was dried and imaged using a phosphoimager. approximately 1.5 x 10 6 hela cells were plated per well of a 6-well dish and 24 hours later incubated with phosphate free media (gibco, 11971-025) for 1 h. thirty minutes before infection, actinomycin d-mannitol (sigma, #a5156) was added to a final concentration of 5 μg ml -1 . infections were carried out in phosphate free media supplemented with 10 μg ml -1 actinomycin d and 2.5 μm 17-dmag. at 1 hpi cells were washed, fresh medium added, and supplemented 3 h later with [ 32 p]-orthophosphoric acid (perkin elmer, #nex053h) 0.20 μci μl -1 . cells were harvested at 6 hpi in rose lysis buffer, and total rna was extracted using ls trizol. rna was separated on a 6m urea-agarose gel as previously described, and detected using a phosphoimager [81] . vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo the role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly effect of vesicular stomatitis virus matrix protein on transcription directed by host rna polymerases i, ii, and iii vesiculoviral matrix (m) protein occupies nucleic acid binding site at nucleoporin pair (rae1 * nup98) inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 vsv disrupts the rae1/ mrnp41 mrna nuclear export pathway vesicular stomatitis virus infection alters the eif4f translation initiation complex and causes dephosphorylation of the eif4e binding protein 4e-bp1 inhibition of host and viral translation during vesicular stomatitis virus infection. eif2 is responsible for the inhibition of viral but not host translation the mechanism of eukaryotic translation initiation: new insights and challenges quantitative studies of mrna recruitment to the eukaryotic ribosome dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis b and t virions ribonucleic acid synthesis of vesicular stomatitis virus. 3. multiple complementary messenger rna molecules polysomal ribonucleic acid of vesicular stomatitis virus-infected hela cells translation of vesicular stomatitis messenger rna by extracts from mammalian and plant cells in vitro synthesis of methylated messenger rna by the virionassociated rna polymerase of vesicular stomatitis virus translation and identification of the mrna species synthesized in vitro by the virion-associated rna polymerase of vesicular stomatitis virus methylated and blocked 5' termini in vesicular stomatitis virus in vivo mrnas ribonucleic acid synthesis of vesicular stomatitis virus, ii. an rna polymerase in the virion l protein requirement for in vitro rna synthesis by vesicular stomatitis virus transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus rna polymerase amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity a single amino acid change in the l-polymerase protein of vesicular stomatitis virus completely abolishes viral mrna cap methylation a unique strategy for mrna cap methylation used by vesicular stomatitis virus unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping rebinding of transcriptase components (l and ns proteins) to the nucleocapsid template of vesicular stomatitis virus location of the binding domains for the rna polymerase l and the ribonucleocapsid template within different halves of the ns phosphoprotein of vesicular stomatitis virus structure of the vesicular stomatitis virus nucleoprotein-rna complex structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor molecular architecture of the vesicular stomatitis virus rna polymerase critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus synthesis of poly(a) in vitro by purified virions of vesicular stomatitis virus in vitro synthesis of rna that contains polyadenylate by virion-associated rna polymerase of vesicular stomatitis virus site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the l gene mrna aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered l protein cis-acting signals involved in termination of vesicular stomatitis virus mrna synthesis include the conserved auac and the u7 signal for polyadenylation translational control of protein synthesis after infection by vesicular stomatitis virus vesicular stomatitis virus mrna and inhibition of translation of cellular mrna-is there a p function in vesicular stomatitis virus? effect of intracellular vesicular stomatitis virus mrna concentration on the inhibition of host cell protein synthesis complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus complete sequences of the ribosome recognition sites in vesicular stomatitis virus mrnas: recognition by the 40s and 80s complexes translation of mrnas from vesicular stomatitis virus and vaccinia virus is differentially blocked in cells with depletion of eif4gi and/or eif4gii a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas preferential translation of vesicular stomatitis virus mrnas is conferred by transcription from the viral genome high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling a systematic view on influenza induced host shutoff ribosome profiling reveals translational upregulation of cellular oxidative phosphorylation mrnas during vaccinia virus-induced host shutoff localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription transcriptional mapping of vesicular stomatitis virus in vivo order of transcription of genes of vesicular stomatitis virus determination of molar ratios of vesicular stomatitis virus induced rna species in bhk21 cells genome-wide determination of rna stability reveals hundreds of short-lived noncoding transcripts in mammals tail-seq: genome-wide determination of poly(a) tail length and 3' end modifications antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus hsp90 chaperoning in addition to phosphoprotein required for folding but not for supporting enzymatic activities of measles and nipah virus l polymerases inhibition of heat shock protein hsp90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation crystal structure and molecular modeling of 17-dmag in complex with human hsp90 txnip potentiates redd1-induced mtor suppression through stabilization of redd1 chemical inhibition of rna viruses reveals redd1 as a host defense factor apobec3g-regulated host factors interfere with measles virus replication: role of redd1 and mammalian torc1 inhibition separate pathways of maturation of the major structural proteins of vesicular stomatitis virus biophysical studies of eif4e cap-binding protein: recognition of mrna 5' cap structure and synthetic fragments of eif4g and 4e-bp1 proteins an overlapping protein-coding region in influenza a virus segment 3 modulates the host response selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein characterization of a second vaccinia virus mrna-decapping enzyme conserved in poxviruses vaccinia virus d10 protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression malignant transformation by a eukaryotic initiation factor subunit that binds to mrna 5' cap new mrnas are preferentially translated during vesicular stomatitis virus infection pathway of vesicular stomatitis virus entry leading to infection entry pathway of vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization systematic identification of type i and type ii interferoninduced antiviral factors eif3 targets cell-proliferation messenger rnas for translational activation or repression 5' utr m(6)a promotes cap-independent translation eif3d is an mrna cap-binding protein that is required for specialized translation initiation dynamic m(6)a mrna methylation directs translational control of heat shock response ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination we thank members of the whelan lab for scientific discussions and suggestions; we further thank carl novina and cailin joyce for help with polysome profiling, keith ketterer for assistance with clc genomics workbench, and alos diallo for help with r and statistical analyses. we thank david stein and hong moulton, of oregon state university, for design and production of the ppmos used in this study. key: cord-318587-ewvnkdr2 authors: steeds, kimberley; hall, yper; slack, gillian s.; longet, stephanie; strecker, thomas; fehling, sarah katharina; wright, edward; bore, joseph akoi; koundouno, fara raymond; konde, mandy kader; hewson, roger; hiscox, julian a.; pollakis, georgios; carroll, miles w. title: pseudotyping of vsv with ebola virus glycoprotein is superior to hiv-1 for the assessment of neutralising antibodies date: 2020-08-31 journal: sci rep doi: 10.1038/s41598-020-71225-1 sha: doc_id: 318587 cord_uid: ewvnkdr2 ebola virus (ebov) is an enveloped, single-stranded rna virus that can cause ebola virus disease (evd). it is thought that evd survivors are protected against subsequent infection with ebov and that neutralising antibodies to the viral surface glycoprotein (gp) are potential correlates of protection. serological studies are vital to assess neutralising antibodies targeted to ebov gp; however, handling of ebov is limited to containment level 4 laboratories. pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio-containment, in serological and viral entry assays. however, neutralisation capacity can differ among pseudotyped virus platforms. we evaluated the suitability of ebov gp pseudotyped human immunodeficiency virus type 1 (hiv-1) and vesicular stomatitis virus (vsv) to measure the neutralising ability of plasma from evd survivors, when compared to results from a live ebov neutralisation assay. the sensitivity, specificity and correlation with live ebov neutralisation were greater for the vsv-based pseudotyped virus system, which is particularly important when evaluating ebov vaccine responses and immuno-therapeutics. therefore, the ebov gp pseudotyped vsv neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against ebov. www.nature.com/scientificreports/ and time required for plaque development, which can take approximately nine days, makes it time-consuming and restricts high-throughput sample processing. development of novel serological assays that utilise genetically modified recombinant or chimeric viruses with attenuated pathogenicity have enabled more widespread investigation of neutralising antibodies against highly pathogenic viruses including ebov 10, 11 . pseudotyped viruses are replication-defective chimeric virions that comprise the structural and enzymatic core of one virus, bearing the envelope protein or glycoprotein of another, and encode a quantifiable reporter gene. retroviruses, including lentiviruses and gammaretroviruses such as human immunodeficiency virus (hiv) and murine leukaemia virus (mlv), respectively, and rhabdoviruses, such as vesicular stomatitis virus (vsv), have been used extensively as cores for pseudotyped viruses 12, 13 , including for ebov 14, 15 . a number of ebov gp pseudotyped virus neutralisation assays have been developed to investigate immune responses to ebov infection and vaccination [16] [17] [18] , as well as for evaluation of monoclonal antibody (mab) therapies [19] [20] [21] . there are many factors that need to be considered when developing and optimising pseudotyped virus neutralisation assays, to assess experimental parameters that can affect assay performance and to ensure accuracy and reproducibility. these include, choice of core virus and reporter gene, determination of target cell line and amount of pseudotyped virus input, as well as correlation with live virus neutralisation 22 . the aim of this study was to assess the suitability of ebov gp pseudotyped hiv-1 and vsv systems to measure neutralisation by evd survivor plasma, in comparison with results from a live ebov neutralisation assay. cell tropism of ebov gp pseudotyped viruses. pseudotyped hiv-1 and vsv bearing the envelope gp from ebov (mayinga) were generated and quantified by measuring luminescence in a range of target cell lines, in order to determine the optimum cell line to use in neutralisation assays. cells only controls were used to determine background levels of luminescence ( supplementary fig. s1 ). reporter activity was detected in all cell lines infected with ebov gp pseudotyped hiv-1 and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed (fig. 1a,b) . for ebov gp pseudotyped hiv-1, highest tcid 50 /ml values were observed in 293t/17 cells, followed by huh-7 cells (fig. 1c) . titres generated by infection of 293t/17 cells were approximately 3, 33 and 73 times greater than those produced by infection of huh-7, hela and vero e6 cells, respectively. for ebov gp pseudotyped vsv, highest titres were obtained in during the initial stages of assay development, it is important to evaluate neutralisation of pseudotyped viruses using well characterised antibodies in order to demonstrate the validity and accuracy of the assay. the ebov gp pseudotyped viruses were assessed for neutralisation by the human anti-ebov gp mab, kz52. kz52 is an antibody isolated from a human survivor of the 1995 outbreak in kikwit that neutralises ebov in vitro and recognises a conformational epitope at the base of the gp [23] [24] [25] . human anti-ebov gp mab, kz52 was unable to neutralise the ebov gp pseudotyped hiv-1 (fig. 2a) within the range tested, however it was able to neutralise the ebov gp pseudotyped vsv (fig. 2b) , suggesting that vsv-based pseudotyped viruses are more sensitive to neutralisation then lentiviral-based, possibly the density of ebov gp on the pseudotyped hiv-1 may differ from that on the pseudotyped vsv or live ebov. to determine the optimal pseudotyped virus input to use in the hiv-and vsv-based assays, neutralisation of different amounts of the ebov gp pseudotyped viruses by plasma from a guinean evd survivor donor or human anti-ebov gp mab kz52 was assessed. kz52 was selected as it is commercially available and there is accompanying information regarding its neutralisation activity against ebov gp pseudotyped vsv expressing luciferase. however, as the ebov gp pseudotyped hiv-1 was not neutralised by kz52 (fig. 2a) in the range tested, plasma from an evd survivor was used to assess the effect of pseudotyped hiv-1 input on neutralisation instead. survivor plasma sample cs001 was chosen as it displayed strong neutralising ability against live ebov neutralisation, with a geometric mean titre (gmt) of 1,218. percentage infectivity was determined relative to infectivity of cells by the ebov gp pseudotyped viruses alone (fig. 3a ,b) and 50% inhibitory concentration (ic 50 ) of pseudotyped virus neutralisation were estimated by model of nonlinear regression dose-response curves (fig. 3c,d) . plasma from evd survivor cs001 displayed neutralising activity against all amounts of ebov gp pseudotyped hiv-1 tested (fig. 3a) . lower pseudotyped virus input resulted in larger variability and less curve fitting. therefore, an ebov gp pseudotyped hiv-1 input of at least 8.6 × 10 4 rlu/well, with a target input of 2.0 × 10 5 rlu/well, was used in subsequent neutralisation assays. kz52 neutralised all dilutions of ebov gp pseudotyped vsv tested (fig. 3b) and ic 50 values decreased with decreasing amounts of pseudotyped virus input (fig. 3d , supplementary table s1 ). when using 3.9 × 10 4 rlu/well of ebov gp pseudotyped vsv, ic 50 of virus neutralisation (0.07 µg/ ml) was similar to that expected according to the manufacturer's product data sheet (0.06 µg/ml). therefore, a target input of approximately 3.7 × 10 4 rlu/well was used in subsequent ebov gp pseudotyped vsv neutralisation assays. table s2 ). neutralisation of ebov gp pseudotyped hiv-1 and vsv by positive (evd survivor) and negative (uk donor) control plasma was assessed in several independent assays ( supplementary fig. s2 ). the background level of neutralisation was determined using plasma from a uk negative control donor. for the hiv-1-based assay this was calculated as ic 50 6.28 reciprocal dilution. the negative control plasma displayed no neutralising activity against ebov gp pseudotyped vsv, and therefore the background level of neutralisation was assigned the lowwww.nature.com/scientificreports/ est dilution of sample tested in the assay (1/20). in the hiv-1-based assay, dose-response curves were unable to be fitted for three of the 30 evd survivor samples and six of the samples were deemed below the background level of neutralisation. in contrast, a dose-response curve was unable to be fitted for only one of the evd survivor samples tested in the vsv-based neutralisation assay. in the hiv-1-based assay, three of the 10 negative plasma samples tested were above the background level of neutralisation, whereas only one of the negative samples tested was above the background level of neutralisation in the vsv-based assay. although some differences in the discriminatory power of positive and negative samples between the assays were observed, a statistically significant difference in neutralisation titres was detected between the evd survivors and negative plasma samples in the hiv-1-based assay (mann-whitney, p = 0.0054) (fig. 4a ) and in the vsv-based assay (mann-whitney, p < 0.0001) (fig. 4b , supplementary table s3 ). remarkably, this difference was more significant and the separation of the positive and negative plasma was better in the vsv-based assay (fig. 4b) . the sum of these results clearly show that the vsv-based ebov gp neutralisation assay displayed better reliability, specificity and sensitivity compared to the hiv-1-based assay. correlation with live ebov neutralisation. the neutralising capacity of the individual plasma samples against authentic ebov was assessed by a live virus neutralisation assay (supplementary table s3 , supplementary fig. s3 ). when ic 50 values of ebov gp pseudotyped hiv-1 neutralisation of the 30 evd survivor and 10 negative plasma samples were compared with gmt values for the live ebov neutralisation assay, a positive correlation (r s = 0.54) was determined using the nonparametric spearman correlation coefficient (fig. 5a) and this was statistically significant (p = 0.0004). remarkably, a stronger statistically significant (p < 0.0001) positive correlation (r s = 0.86) was observed when ic 50 values of ebov gp pseudotyped vsv neutralisation were compared with gmt values for the live ebov neutralisation assay (fig. 5b) . the correlation coefficients for ebov gp hiv-1 and vsv ic 50 compared with live ebov gmt without the negative controls were 0.38 (p = 0.0375) and 0.69 (p < 0.0001), respectively. therefore, the vsv-based ebov gp pseudotyped virus neutralisation assay correlated better with live ebov neutralisation than the hiv-1-based neutralisation assay. pseudotyped viruses can be used as alternatives to infectious virus in serological assays to measure neutralising antibodies to viral envelope glycoproteins 11 . pseudotyped virus assays used to profile neutralising antibody responses against severe acute respiratory syndrome-associated coronavirus (sars-cov) 26 , influenza (h5n1 and h7n9) [27] [28] [29] , rabies 30,31 and chikungunya virus 32 , for example, found that results correlated well with those from replication-competent or live virus assays. a high degree of correlation has been demonstrated between ebov cl4 prnt and an ebov pseudotyped vsv cl2 fluorescence reduction neutralisation test (frnt) 33 . however, pseudotyped virus assays may not always accurately determine neutralisation 34, 35 . live ebov and ebov gp pseudotyped neutralisation assays have previously been shown to yield variable results 8, 36 , which could be due to differing experimental conditions and viral systems. it is therefore important to optimise pseudotyped virus neutralisation assays in context of the particular viral gp being studied in order to obtain reliable specificity and sensitivity. the aim of this study was to assess the suitability of ebov gp pseudotyped hiv-1 and vsv systems to measure the neutralising ability of plasma from evd survivors, when compared to live ebov neutralisation. reporter activity was detected in all cell lines (293t/17, huh-7, hela and vero e6) infected with ebov gp (mayinga) pseudotyped hiv-1 and vsv, demonstrating the broad tissue range conferred by ebov gp, although differences in luminescence were observed. this may reflect general defects in viral entry in different cells. a relatively lower level of ebov gp pseudotyped hiv-1 transduction was exhibited by vero e6 cells, which might be due to an intrinsic restriction factor, trim5α, which restricts retroviral infection by specifically recognising the hiv-1 capsid and promoting its rapid, premature disassembly 37 . highest tcid 50 values were obtained following ebov gp pseudotyped hiv-1 and vsv infection of 293t/17 and vero e6 cells, respectively. there seemed to be large variability of the luminescent measurement for the vsv-based platform, which may be caused by the www.nature.com/scientificreports/ sensitive nature of the luciferase signal detection. this highlights the importance of titrating each pseudotyped virus batch before use in neutralisation assays, and the inclusion of multiple replicates. the ebov gp pseudotyped viruses were used to assess the neutralising activity of a human anti-ebov gp mab, kz52. kz52 has been shown previously to neutralise ebov pseudotyped viruses 17, 19, 38 . however, within the range tested here, kz52 did not display neutralisation against ebov gp pseudotyped hiv-1, suggesting that the ebov gp on the pseudotyped hiv-1 might be at higher levels, thereby reducing assay sensitivity, and neutralisation may be observed using a higher concentration of kz52. in contrast, kz52 was able to neutralise the ebov gp pseudotyped vsv. to assess the effects of differing amounts of pseudotyped virus input on neutralisation, plasma from an evd survivor of the 2013-2016 ebov outbreak and kz52 were screened against different amounts of the ebov gp pseudotyped hiv-1 and vsv, respectively. decreasing quantities of pseudotyped hiv-1 led to more variable and unreliable results, and the kz52 ic 50 of pseudotyped virus neutralisation decreased with decreasing amounts of ebov gp pseudotyped vsv input. the variability in neutralisation observed between different amounts of pseudotyped virus input highlights the importance of including standards or reference material with a known activity or potency when comparing neutralising activity, allowing calibration of results 39 . both pseudotyped virus systems were able to measure neutralising antibodies in plasma from evd convalescent patients, and results correlated positively with a live ebov neutralisation assay. however, the discriminatory power of the hiv-1-based assay with regards to differing antibody titres appeared to be low. some of the samples tested, which showed neutralising activity against live ebov, did not display neutralisation against the pseudotyped virus and vice versa, therefore raising questions on the sensitivity and specificity of the pseudotyped hiv-1 assay. in the current study, human embryonic kidney (293t/17) cells were used for the pseudotyped hiv-1 neutralisation assays, whereas african green monkey kidney (vero) cells were used in the vsv-based assay and also the live ebov assay. therefore, this could account for some of the differences in results observed between the two assays and for the better performance of the vsv-based assay in relation to live ebov neutralisation. also, the hiv-1-and vsv-based pseudotyped virus systems assessed in the current study utilise different transfection methods, which could have implications on the composition of the pseudotyped viruses, density and/or glycosylation of the viral envelope protein on the surface, and consequently neutralisation results. this highlights the importance of assessing experimental conditions and methodology when developing and optimising pseudotyped virus neutralisation assays. a limitation to this study was that the level of ebov gp incorporation per pseudotyped virus type could not be assessed. also, for the vsv-based pseudotyped virus system, traces of vsv-g from the rvsv-δg-luc-vsv-g virus could be recycled into newly pseudotyped virions 40 . therefore, the use of anti-vsv-g hybridoma cell culture supernatant could give rise to pseudotyped virions covered by anti-vsv-g antibodies, but are still infectious due to ebola gp. this could potentially induce plasma specific reactivity of virions due to bound anti-vsv-g antibodies more than ebov gp specific reactivity. there are several differences between ebov gp pseudotyped and live ebov neutralisation assays that could affect their results 8 . due to their non-replicating nature, such pseudotype systems do not recapitulate all steps in the viral life cycle that may potentially be targeted by neutralising antibodies 41 . in addition, the round, spherical shape of ebov gp pseudotyped hiv-1 or bullet shape of ebov gp pseudotyped vsv compared to the filamentous shape of authentic ebov could affect their susceptibility to neutralisation. also, the density of gp on the surface of the pseudotyped virus may not be the same as that found on live ebov and may result in the loss or masking of quaternary epitopes 11, 42 . furthermore, gp maturation and assembly in live ebov could be different in the generation of an ebov pseudotyped virus and may result in different targets and/or conformational epitopes when using whole live ebov as opposed to ebov gp alone in a pseudotyped virus. the presence of shed gp or secreted gp (sgp) in the live ebov assay compared to absence in the ebov gp pseudotyped virus assays could also have an effect on neutralisation. in the live ebov assay, shed gp and sgp could reduce neutralisation of circulating virus by cross-reactive antibodies to surface gp. however, in the current study, weaker relative neutralisation was observed in the hiv-1 based pseudotyped virus assay. therefore, it is possible that cell debris or free gp generated during ebov gp pseudotyped hiv-1 production by polyethylenimine (pei) transfection could be interfering with neutralisation. finally, detection of infected cells via measurement of luminescence in the ebov gp pseudotyped virus neutralisation assay compared to plaque formation in the live ebov neutralisation assay could affect neutralisation readout. ebov gp pseudotyped virus neutralisation assays have value for vaccine evaluation and assessment of convalescent blood products and mabs for use as immunotherapeutics. however, pseudotyped virus assays may not always accurately determine neutralisation when compared with neutralisation against live virus. in this study, both ebov gp pseudotyped hiv-1 and vsv assays were able to detect neutralisation of plasma from evd survivors and correlated positively with live ebov neutralisation. however, the vsv-based assay performed better than the hiv-1-based assay in relation to specificity, sensitivity, and correlation with the live ebov neutralisation assay. this research has highlighted the importance of optimising pseudotyped virus neutralisation assays in context of the particular viral gp being studied, especially when evaluating vaccine responses and therapeutics, and could provide a better understanding of the correlates of protection against ebov. and from negative control blood donors in the uk and guinea, who were not knowingly exposed to persons with evd and did not attend high risk events such as funerals, were heat inactivated at 56 °c for 30 min. the samples were obtained from a pre-existing biobank, for which live ebov neutralisation 45 production of pseudotyped viruses. the generation of hiv-1 pseudotyped viruses was performed as detailed previously 44, 46, 47 . twenty-four hours prior to transfection, approximately 8 × 10 5 293t/17 cells were seeded into sterile, 6-well cell culture plates (corning, ewloe, uk) and incubated at 37 °c, 5% co 2 and 95% humidity until 60-80% confluence. the hiv gag-pol plasmid, p8.91, and the firefly luciferase reporter construct, pcsflw, were transfected simultaneously with the ebov (mayinga) gp expression vector at a ratio of 0.6:0.9:0.6 µg (core:reporter:envelope) using 10 µl of 1 µg/ml polyethylenimine (pei) (sigma-aldrich) per 1 µg dna in opti-mem medium (gibco). following overnight transfection, the cells were incubated with fresh medium and incubated at 37 °c, 5% co 2 . pseudotyped virus supernatants were harvested at 48 and 72 h posttransfection, passed through a 0.45 µm pore filter (millex, millipore, watford, uk) and stored at − 80 °c. ebov gp pseudotyped vsvs were prepared using recombinant vsv, in which the vsv-g gene had been deleted (rvsv-δg) and replaced with a luciferase reporter gene (rvsv-δg-luc) by a method similar to that described previously 48 . twenty-four hours prior to transfection, approximately 2.4 × 10 6 293t/17 cells were seeded into sterile, 100 mm cell culture dishes (corning) and incubated at 37 °c, 5% co 2 and 95% humidity until 60-80% confluence. the cells were transfected with the ebov gp expression vectors using transit-lt1 transfection reagent (mirus bio, madison, wisconsin (wi), usa) as per the manufacturer's instructions. following overnight transfection, the medium was removed and the cells were infected with rvsv-δg-luc that was pseudotyped with the vsv glycoprotein (rvsv-δg-luc-vsv-g) (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a multiplicity of infection (moi) of 5 in opti-mem medium and incubated at 37 °c, 5% co 2 . after 2 h, the inoculum was removed, cells were washed twice with dulbecco's phosphate buffered saline (dpbs) (gibco) and fresh medium was added. pseudotyped virus supernatants were harvested at 18-24 h post-infection, clarified twice by centrifugation at 200xg for 5 min at 10 °c and stored at − 80 °c. prior to use, the pseudotyped viruses were incubated with anti-vsv-g hybridoma cell culture supernatant (masayuki saijo, national institute of infectious diseases, tokyo, japan) at a 1:125 dilution for 1 h at 37 °c to reduce background infection mediated by residual virus possessing vsv-g, which can be carried over during preparation 48 . all experiments involving pseudotyped viruses were performed in a cl2 facility at public health england (phe), porton down, uk. pseudotyped virus titration and neutralisation assays. titration and neutralisation assays were performed in 96-well solid white flat bottom polystyrene tc-treated microplates (corning) and were based upon previously described protocols 44, 46, 48 . for pseudotyped hiv-1 titration assays, five-fold serial dilutions of pseudotyped virus at a starting dilution of 1:5 were prepared in quadruplicate in opti-mem medium at a final volume of 100 µl/well. 100 µl of approximately 2 × 10 4 293t/17, huh-7 or vero e6 cells, or 1 × 10 4 hela cells were then added to each well and incubated at 37 °c, 5% co 2 for 48 h. the medium was removed and 50 µl of a 50:50 mix of bright-glo luciferase assay reagent (promega, southampton, uk):fresh medium was added to each well and incubated for at least 2 min at room temperature to allow complete cell lysis. luminescence was measured using a glomax-multi + detection system luminometer (promega) and relative luminescence units per ml (rlu/ml) were determined. the negative cut-off was set at 2.5 times the average rlus of the cells only control wells. 50% tissue culture infectious dose (tcid 50 )/ml values were determined using the reed-muench method 49 . for the pseudotyped hiv-1 neutralisation assay, two or threefold serial dilutions of plasma samples at a starting dilution of 1:5 or 1:10, respectively, were prepared in duplicate in opti-mem medium at a final volume of 50 µl/well and incubated with 50 µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for 1 h at 37 °c. 100 µl of approximately 2 × 10 5 293t/17 cells were then added to each well and incubated for 48 h at 37 °c, 5% co 2 , prior to taking a chemiluminescent readout as described above. infectivity was calculated using the formula: percentage (%) infectivity = [(rlu with sample)/(rlu without sample)] × 100. www.nature.com/scientificreports/ for pseudotyped vsv titration assays, 24 h prior, approximately 2.5 × 10 4 293t/17 or 1 × 10 4 huh-7, hela cells or vero e6 cells were seeded in 96-well microplates and incubated at 37 °c, 5% co 2 and 95% humidity. the medium was removed and two-fold serial dilutions of pseudotyped virus in opti-mem medium, starting with neat pseudotyped virus were added to each well in quadruplicate at a final volume of 100 µl/well. after 24 h, a chemiluminescent readout was taken and tcid 50 /ml values were determined as described above. twenty-four hours prior to pseudotyped vsv neutralisation, approximately 1 × 10 4 vero e6 cells were seeded and incubated as for titration above. twofold serial dilutions of plasma samples at a starting dilution of 1:10 were prepared in duplicate in opti-mem medium at a final volume of 120 µl/well in 96-well microplates, and incubated with 120 µl of a standardised rlu per well of pseudotyped virus (as calculated from the titration assay), prepared in opti-mem medium, for 1 h at 37 °c. the medium was removed from the cells, 50 µl of the plasma-pseudotyped virus mixtures were added to each well in quadruplicate at incubated at 37 °c, 5% co 2 . after 1 h, 50 µl of fresh medium was added to each well. luminescence was measured after 24 h and infectivity was calculated as described above. statistical analysis. pseudotyped virus neutralisation assay raw data were normalised as percentage (%) infection relative to mean values for pseudotyped virus only controls (equivalent to 100% infection), then ic 50 of pseudotyped virus neutralisation were estimated by model of nonlinear regression fit with settings for log (inhibitor) vs. normalised response curves using graphpad prism v5 (san diego, california (ca), usa). statistical comparison between two unpaired groups was performed using the mann-whitney test (graph-pad prism v5). correlation between two variables was quantified using spearman nonparametric correlation (graphpad prism v5). www.nature.com/scientificreports/ open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/. ebola haemorrhagic fever emergence of zaire ebola virus disease in guinea world health organization. situation report -10 biochemical analysis of the secreted and virion glycoproteins of ebola virus a mutation in the ebola virus envelope glycoprotein restricts viral entry in a host species-and cell-type-specific manner characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms systematic analysis of monoclonal antibodies against ebola virus gp defines features that contribute to protection role of antibodies in protection against ebola virus in nonhuman primates immunized with three vaccine platforms the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination current progress with serological assays for exotic emerging/re-emerging viruses construction and use of a human immunodeficiency virus vector for analysis of virus infectivity a system for functional analysis of ebola virus glycoprotein distinct mechanisms of entry by envelope glycoproteins of marburg and ebola (zaire) viruses identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps specific neutralizing response in plasma from convalescent patients of ebola virus disease against the west africa makona variant of ebola virus ebola virus neutralizing antibodies detectable in survivors of the yambuku, zaire outbreak 40 years after infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb-003 cocktail antibodies protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody potent neutralizing monoclonal antibodies against ebola virus infection technical considerations for the generation of novel pseudotyped viruses ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody structure of the ebola virus glycoprotein bound to an antibody from a human survivor longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h5n1 influenza viruses characterization of lentiviral pseudotypes with influenza h5n1 hemagglutinin and their performance in neutralization assays safe pseudovirus-based assay for neutralization antibodies against influenza a(h7n9) virus a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system development of a pseudotyped-lentiviral-vector-based neutralization assay for chikungunya virus infection high degree of correlation between ebola virus bsl-4 neutralization assays and pseudotyped vsv bsl-2 fluorescence reduction neutralization test human immunodeficiency virus type 1 env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies broadly neutralizing human monoclonal antibodies to the hepatitis c virus e2 glycoprotein comparison of platform technologies for assaying antibody to ebola virus specific recognition and accelerated uncoating of retroviral capsids by the trim5α restriction factor a shared structural solution for neutralizing ebola viruses ebola virus: pseudotypes, libraries and standards characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins neutralizing antibodies inhibit chikungunya virus budding at the plasma membrane maturation of west nile virus modulates sensitivity to antibody-mediated neutralization multiply attenuated lentiviral vector achieves efficient gene delivery in vivo investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison phase 1 trials of rvsv ebola vaccine in africa and europe a sensitive retroviral pseudotype assay for influenza h5n1-neutralizing antibodies lyophilisation of influenza, rabies and marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit generation of vsv pseudotypes using recombinant δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines a simple method of estimating fifty per cent endpoints the authors would like to thank masayuki saijo for providing the rvsv-δg-luc-vsv-g virus and anti-vsv-g hybridoma cell culture supernatant. we are grateful to eccac for providing vero e6 and hela cells, and to arvind patel for providing huh-7 cells. this work was funded by the u.s. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-71225 -1.correspondence and requests for materials should be addressed to m.w.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-104239-xxlcdbqi authors: nan title: the organization of endoplasmic reticulum export complexes date: 1996-10-01 journal: j cell biol doi: nan sha: doc_id: 104239 cord_uid: xxlcdbqi export of cargo from the er occurs through the formation of 60-70nm copii-coated vesicular carriers. we have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of er export sites in vivo and in vitro. we find that er buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. the basic organization of an export complex can be divided into an active copii-containing budding zone on a single er cisterna, which is adjacent to budding zones found on distantly connected er cisternae. these budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain copi coats involved in retrograde transport. vesicles within these export complexes contain concentrated cargo molecules. the structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. we conclude that budding from the er through recruitment of copii is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by copi. abstract. export of cargo from the er occurs through the formation of 60-70-nm copii-coated vesicular carriers. we have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of er export sites in vivo and in vitro. we find that er buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. the basic organization of an export complex can be divided into an active copiicontaining budding zone on a single er cisterna, which is adjacent to budding zones found on distantly connected er cisternae. these budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain copi coats involved in retrograde transport. vesicles within these export complexes contain concentrated cargo molecules. the structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. we conclude that budding from the er through recruitment of copi! is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by copi. xport of protein from the er is the first step in the vectorial movement of cargo through compartments of the secretory pathway of eukaryotic cells. pioneering studies by palade (1975) established that the site of exit from the rer in pancreatic acinar cells is through transitional elements, a region of partly rough, partly smooth tubular er juxtaposed to the cis face of the golgi stack. morphologically, er-derived vesicles are 60-70 nm in diameter and contain an electron-dense coat when viewed using transmission electron microscopy (ziegel and dalton, 1962) . this characteristic coat contains copii components that are assembled in response to the activation of the sarl gtpase, a machinery now recognized to be evolutionarily conserved in yeast and mammalian cells (for review see barlowe, 1995) . while the formation of er to golgi carrier vesicles in secretory tissues is largely confined to the transitional region facing the juxtanuclear golgi apparatus (palade, 1975) , studies in other cell lines have shown that export from the er can originate from multiple sites that appear randomly distributed throughout the cytoplasm and, in most instances, distant from the golgi complex. the relationship of these peripheral sites to the transitional region found in secretory cells is unknown, although they are now recognized to consist of clusters of small vesicles and tubular elements (saraste and kuismanen, 1984; schweizer et al., 1990; saraste and svensson, 1991; lotti et al., 1992) re-ferred to as vesicular tubular clusters (vtcs) 1 . while vtcs are readily detectable at 37°c in vivo (saraste and kuismanen, 1984; saraste and svensson, 1991) and at 32°c in vitro (plutner et al., 1992; pind et al., 1994a) , visualization of these structures can be markedly enhanced by incubation of cells at reduced temperature (15°-16°c) (saraste and kuismanen, 1984) , presumably due to a rate-limiting step in membrane flow through these intermediates. elements of vtcs lack luminal continuity with the er (saraste and svensson, 1991; balch et al., 1994; connolly et al., 1994) , although tubular extensions of er into these structures have been observed (stinchcombe et al., 1995) , particularly in cells infected with certain viruses (tooze et al., 1984; krijnse-locker et al., 1994) , reinforcing their close relationship to er export. vtcs are dynamic structures with varied morphology in different cell types. in the past, vtcs have been suggested to be the site of o-glycosylation (tooze et al., 1988) , acylation (rizzolo et al., 1985) , and generation of the mannose-6-phosphate signal for lysosomal protein targeting (pelham, 1988) . several endogenous proteins serve as useful markers for vtcs. these include the small gtpase rab2 (chavrier et al., 1990) , the transmembrane protein p58 in rat cells or its human homologue p53 (schweizer et al., 1988; saraste and svensson, 1991) , which actively cycle between the er and vtcs, and the copi subunit 13-cop (oprins et al., 1993; pepperkok et al., 1993; pind et al., 1994a; aridor et al., 1995) . vtcs play a pivotal role in the segregation of anterograde and retrograde transported proteins (aridor et al., 1995; tang et al., 1995) . segregation is believed to involve the copi coat complex whose assembly is driven through activation of the arf1 gtpase (aridor et al., 1995; letourneur et al., 1995) . while vtc composition and function have been described qualitatively through use of immunofluorescence (lotti et al., 1992; aridor et al., 1995; lippincott-schwartz et al., 1995) and immunoperoxidase (connolly et al., 1994; stinchcombe et al., 1995) approaches, the topology of er budding sites and, in particular, their relationship to vtcs have not been studied quantitatively at high resolution. in the present paper, we use both transmission tem and immunoelectron microscopy in conjunction with quick-freeze, deep-etch methods to reconstruct these relationships morphometrically and to confirm that cargo is concentrated during vesicle budding. we find that almost all er buds detected in the cell were concentrated in regional loci facing vtcs, which had a characteristic diameter and vesicular-tubular composition. as such, the topological organization of buds and vtcs defines a morphological unit of function, which we refer to as export complexes. we conclude that budding from the er is not evenly distributed along the membrane, but is confined to specific export sites. these are highly enriched in copii and copi transport components and are likely to promote the coupling of anterograde and retrograde transport. a polyclonal antibody specific for yeast sec23p that cross-reacts with a mammalian homologue (orci et al., 1991) was obtained from r. schekman (university of california, berkeley). affinity-purified antibodies specific for yeast secl3p that cross-reacts with a mammalian homologue (shaywitz et al., 1995) were a generous gift of c. kaiser (massachusetts institute of technology, cambridge, ma). a hybridoma cell line expressing an mab specific for the carboxyl terminus of vesicular stomafitis virus glycoprotein (vsv-g) (p5d4) was provided by t. kreis (university of geneva, switzerland) (kreis, 1986) . secondary antibodies were obtained from the following sources: texas red--conjugated goat anti-mouse igg from zymed laboratories (south san francisco, ca), 6 nm gold--conjugated goat anti-mouse and anti-rabbit antibodies from jackson immunoresearch laboratories (west groven, pa), and 10 nm gold--conjugated goat anti-mouse antibodies from amersham corp. (arlington heights, il). sarl wild type and the h79g and t39n mutant proteins were prepared as described (aridor et al., 1995) . all other reagents, except where indicated, were obtained from sigma chemical co. (st. louis, mo). normal rat kidney (nrk) and rat basophilic leukemia (rbl) cells (rbl-2h3) were maintained in monolayer culture in a-mem supplemented with penicillin, streptomycin, and 7% (nrk) or 16% (rbl) fbs (gemini bioproducts, calabasas, ca) as described (plutner eta] ., 1992). ts045-vsv (lafay, 1974) was propagated in bhk-21 ceils as described (beckers et al., 1987) . cells were infected with ts045-vsv at a multiplicity of 10-20 plaque-forming units per cell as described (davidson and balch, 1993) . cells were fixed in 2.5% glutaraldehyde (ga) in pbs for 1 h at room temperature (rt), scraped in ga, and pelleted at 15,000 g in a microcentrifuge for 10 rain. the tight pellet was washed in veronal-acetate buffer (ph 6.0) and stained in 1% buffered oso4 for 1 h at rt. after washing in veronalacetate buffer, pellets were stained en bloc with 2% uranyl acetate in veronal-acetate buffer, dehydrated in alcohol and acetone, and embedded in epon 812 (electron microscopy, sciences, fort washington, pa) . for serial sectioning, the cell pellet was cut with a glass knife followed by trimming of the resulting block to obtain an ~40 ×300 ~xm pyramid. a ribbon of 25-35 consecutive sections of 65 nm in thickness was cut with a diamond knife on reichert ultramicrotome 2e and transferred to a single 2 × 1.5 mm slot grid (electron microscopy sciences) precoated with a formvar/carbon film. sections were counterstained with a saturated solution of uranyl acetate in methanol for 10 rain at rt and reynold's lead citrate for 10 min. budding structures on the er and vtcs were followed in consecutive sections, and images were overlaid to reconstruct continuity between structures. stereological parameters. an estimation of the mean volume of the cells was performed by two independent methods (baddeley et al., 1985; griffiths et al., 1989) . in the first case, rbl cells were grown on glass coverslips. cells were fixed with 2.5% ga in pbs, ph 7.4, overnight. the cells were then viewed and photographed under phase contrast with an axiophot (zeiss, oberkochen, germany) with a ×20 objective lens. the images were enlarged photographically 10 times, and the mean surface of the cell projection was determined by the point-counting method (weibel, 1979) using a 5-ram square lattice grid. approximately 10,000 rbl cells were used for the estimation of the mean surface of the cell projection onto a planar surface. the cells on coverslips were stained and embedded in epon as described above. after polymerization, the coverslips were detached from the resin by plunging into liquid nitrogen. thin-strips of embedded cells in resin were then combined face to face and reembedded on top of an epon block to produce "vertical" sections (griffiths et al., 1984) . images of cells were taken with a transmission electron microscope (1200ex; jeol usa, peabody, ma) and photoenlarged (×3,500 total magnification), and the mean height of ~250 cells in a monolayer was measured by the point-counting method (weibel, 1979) . the mean cell volume (vc.r.) was then determined according to formula: where sc.p. is the mean surface of the cell projection (determined by the phase-contrast light microscopy), and hc. is the mean height of the cell (determined by tem). for estimation of the mean cell volume using the second method, nrk and rbl cells were grown on 35-mm tissue-culture dishes (costar corp., cambridge, ma), fixed in the dish in 2.5% ga in pbs for 1 h at rt, and processed for tem as described above. the cell pellet was cut with a diamond knife on a reichert ultramicrotome 2e. 60-65-nm sections were counterstained as described above. approximately 1,200 rbl and 800 nrk cells were randomly chosen and photoenlarged to a magnification of 14,000. the mean surface of the cells on sections were measured by the point-counting method (weibel, 1979) . the mean cell volume was found according to formula (weibel and gomez, 1962) : where sc.s. is the mean cell surface on thin-section, the surface to volume ratio was found according to formula: where i is the number of intersections on a grid, p is the number of points on the grid, and d is the distance between the points (weibel, 1979) . both procedures yielded nearly identical results for rbl cells, the values obtained from the second procedure are reported in table i . the stereological parameters of nuclei, er, and golgi apparatus were determined using the same sections. randomly taken cell contours were enlarged to a total magnification of 14,000 as described above. using the point-counting method, we determined the nucleus volume to cell volume ratio and a nucleus surface to nucleus volume ratio as described (weibel, 1979) . randomly chosen fields of cells containing er or golgi membranes were magnified to 31,000 to establish the er(golgi) volume to cell cytoplasm volume ratio by the point-counting method (weibel, 1979) . some of these images were photoenlarged to 120,000 to determine the er(golgi) surface to volume ratio. the er(golgi) surface was found by formula 3. corrections of bias due to section thickness were done as described (weibel and paumgartner, 1978) . for the er volume, the correction factor was 0.51 and 0.65 for the er surface. assuming that two-thirds of the golgi complex was composed of cisternae and one-third of tubules, the correction factors for golgi volume and surface were estimated to be 0.6 and 0.64, respectively. estimation of number and deasity of total er-derived buds. to estimate the number er-derived buds, we used two distinct criteria: (a) a bud was considered an elevation on the surface of the er with a width of 60-80 nm and covered with a characteristic coat on the external leaflet of the membrane: and (b) buds had a direct connection with the er membrane and were extruded from the membrane by at least 50% of their circumference. the total number of er buds per cell was determined as described (lucocq et al., 1989) . to obtain this value, we multiplied the total number of er buds detected in thin-secfions of ~300 cells by the mean volume of cells and divided that value by the total volume contributed by the ~300 cells. the latter value was found by multiplying the thickness of a section by the total surface area of the ~300 cell sections in which er buds were counted. by using relatively thick sections corresponding to the average diameter of an er bud and the above two criteria, we were able to avoid double counting most of the er buds present on consecutive thin sections. to establish this point independently, we performed a separate experiment in which the total number of er buds was counted on several stacks of serial thin-sections. by comparing the number of er buds determined by this reconstruction approach to the number determined by counting of individual sections (indirect method), we found that overestimation by the latter technique was <10-15%. hence, both methods could be used interchangeably. we also found that the indirect method showed a high reproducibility. in spite of the high degree of er bud enrichment in local zones, the comparison of estimates of the total number of buds per cells between groups of as few as 35 randomly taken cells with a total surface of section of 2,500 tj.m 2 gave a value of variance of less than 5% from one group to another. in addition, in four different experiments, the value of the total number of buds per cell was found to be the same. because of technical simplicity, we routinely used the indirect method to estimate the average number of buds per cell. buds in the golgiexclusion zone. the golgi exclusion zone is defined as the golgi-containing region found in the pericentrosomal region of rbl cells and includes directly adjacent bud-bearing cisternae of the er. the surface area of the golgi exclusion zone in each individual section within a stack of 11-20 consecutive sections (referred to as a disector [sterio, 1984; lucocq et al 1989] ) was determined by the point-counting method (weibel, 1979) , and the mean surface area in each disector was calculated for 12 different cells (range from 30-90 ~tm2). to find the volume of the golgi exclusion zone included in the disector, the mean surface area of the golgi exclusion zone in each disector was multiplied by the thickness of the section and the number of individual sections (35 to 105 ixm 3 for 12 individual disectors). the total number of er buds within each disector of a given cell was found by reconstruction from serial sections. the volumetric density of er buds was calculated by dividing the number of buds found within each disector by the volume of the disector. the total volume and surface of er membranes in the golgi exclusion zone was found by the point-counting method (weibel, 1979) . by dividing the total number of er buds by the er surface within the golgi exclusion zone, we calculated the density of er buds in this region. buds outside of the golgi exclusion zone. the density of er buds outside of the golgi exclusion zone was found according to the procedure described above for the golgi exclusion zone. buds in export complexes. the local density of buds on individual cisternae associated with export complexes was determined as follows: using a stack of sequential serial sections, we followed one continuous bud-bearing zone of er membrane that contained at least four buds. the distances between the most distant er buds in the stack were directly measured in both the plane of the section and the depth of the stack. these two distances were multiplied by one another to get a surface area of the plane of the er bud-bearing region. the total number of er buds in such a zone (obtained by reconstruction as described above for the golgi exclusion zone) was then divided by the area of the bud-bearing zone to determine a local er bud surface density. es.lmation of number of vtcs. the number of vtcs per cell was determined using the disector method (sterio, 1984) as described (lucocq et al., 1989) . sections of 26 random cells were photographed at a calibrated magnification of 7,000 throughout 25-35 consecutive sections in which they were present. each "end section" was designated the "lookup" section, and all clusters present in the other sections, but not in the look-up section, were counted (q). then the volume of disector (vdi~) was determined by multiplying the average surface of individual cells in the stack found by the point-counting method by the depth of the dissector, which is equal to the thickness of the section multiplied by the number of sections in the disector. the total number of clusters in individual ceils (nclustcrs/cell) was determined by the formula, (4) to determine the number of elements in vtcs, the value reported assumes that all elements found in consecutive sections are discontinuous with one another. this value is likely to be an overestimate, as some of the tubular structures within vtcs may extend across several sections. the number of elements in vtcs was determined by direct counting of individual profiles on each serial section. these were summed throughout all sections through a given vtc to obtain a total value. probability measurements. to estimate the probability of a bud having proximity to a second bud in the cell, images of serial sections were photoenlarged to 40,000 to reconstruct a section of the cell in three dimensions. 300 randomly chosen er buds were taken as the center of reference (referred to as reference buds), and the distance between each individual reference bud and other er buds in the same and consecutive serial sections encompassing up to 1.3 p.m distance above and below the reference bud was measured directly. a series of concentric shells with a volume equal to the volume of the most internal sphere having a 0.2-1xm diam (corresponding to a volume of 0.0042 i~m 3) was constructed around the reference bud. subsequently, each measured bud was assigned to a shell with its distance from the reference bud being that of the corresponding outer diameter of the shell. the probability of finding a bud within a particular shell was determined by dividing the number of positive shells by the number of reference buds, and the value was reported as a percentage. sem. statistical calculations were performed by determining the sem for the pooled stereological data for each condition as described in the results. nrk cells grown on 35-mm tissue-culture dishes were infected with vesicular stomatitis virus (strain ts045), postinfected for 4 h at 39.5°c, and permeabilized as described (plutner et al.. 1992) . after incubation at the permissive temperature (32°c) as described in the results, the cells were fixed for 30 min with 3% paraformaldehyde and 0.1% ga in pbs (ph 7.4), washed for l0 rain in pbs containing 0.05 m glycine, scraped, mixed with a preheated (40°c) 10% gelatin in pbs, and centrifuged at 15,000 g in a microcentrifuge for 10 min. cells embedded in gelatin were cooled on ice, and a solid pellet was cut into i-ram-wide cubes. after overnight cryoprotection by infiltration with a mixture of 2.3 m sucrose in 0.1 m phosphate buffer (ph 7.4) containing 20% polyvinyl pyrrolidene, the cubes were mounted on aluminum nails and frozen in liquid nitrogen. ultrathin cryosections cut on a reichert ultracut e, equipped with a fc-4 cryoattachment, were picked up with 2.0 m sucrose-1% bsa in pbs and collected on formvar/carbon-coated nickel grids. sections were then quenched in 0.01 m glycine in pbs, incubated for 30 min in 10% fbs-pbs at rt, and for 1-2 h with primary antibodies diluted in 10% fbs-pbs antibody. excess primary antibody was removed by multiple rinses in 5% fbs-pbs, followed by transfer of the section to a drop of 10% fbs-pbs containing 6 or 10 nm gold--conjugated anti-rabbit antibodies. after a 2-h incubation at rt, the grids were washed in double-distilled water and stained in 2% neutral uranyl acetate (10 rain), followed by embedment in 3.2% polyvinyl alcohol/0.2% methyl cellulose~ containing 0.2% uranyl acetate. no labeling was observed in controls in which primary antibodies were omitted. nrk cells were infected with ts045 vsv as described above. after digitonin permeabilization (plutner et al., 1992) and incubation in vitro as described in the results, cells were fixed with 0.025% ga/3% paraforrnaldehyde for 30 rain. cells were washed three times with pbs, quenched with 0.05 m glycine in 10% fbs/pbs for 30 rain, and incubated overnight with an anti-vsv-g cytoplasmic tail mab (p5d4) (kreis, 1986) . cells were washed twice with fbs/pbs, followed by incubation with rabbit anti-mouse antibodies for l-2 h and with 6 or 10 nm gold conjugated to anti-rabbit antibodies for 2 h. excess unbound antibodies were removed by washing, and the cells were fixed with 2.5% ga for 30 rain. cells were scraped, pelleted, and processed for epon embedding. for three-dimensional visualization using quick-freeze, deep etch replicas, immunolabeling was performed on glass coverslips. for quantitation, the epon-embedded cell pellet was cut as described above. images (xl0,000) were scanned into a computer, and membrane outlines of the er and vtcs were measured using the program nih image, version 5.9. the linear density of gold particles corresponding to vsv-g was determined as described . for determination of the distribution of vesicles and vtcs, nrk cells grown on glass coverslips were permeabilized and incubated as described in the results, fixed and labeled for vsv-g using the immunodiffusion protocol, and embedded in epon on the glass coverslip as described above. after detachment from glass, thin layers of cells embedded in epon were sandwiched against each other and reembedded in epon. vertical sections of cells were prepared and counterstained as described above. er to golgi intermediates were identified based on either the presence of gold particles corresponding to vsv-g or on their characteristic morphology as described in the results. after incubation in vitro as indicated in the results, semi-intact cells grown on 9 × 9 mm glass coverslips were fixed in 2.5% ga in pbs for 1 h, washed in pbs, and divided into small pieces (~3 mm2). coverslips were rinsed exhaustively in double-distilled water, followed by a rinse with 10% methanol in water, and quick-frozen using a liquid nitrogen-cooled copper block gravity press (hitek, benicia, ca). the cells were fractured with a razor blade under liquid nitrogen, freeze dried in a vacuum evaporator (400; balzers, inc., lichtenstein) and replicated with ~2 nm of platinum that was rotary deposited from 24 ° above the horizontal. the replica was then reinforced with ~140/~ of carbon using an electron gun at an angle of 90 ° to the horizontal. a drop of 2% colloidion solution was applied on the replica membrane. the coverslips were detached in a 40% solution of hydrofluoric acid, and cells were dissolved in chlorox. after washing, replicas were transferred to formvar-coated copper grids, the colloidion film on replicas was dissolved with amylacetate, and images were examined using tem. the basic stereological parameters of the rbl and nrk cell line used in these studies are shown in table i . to morphometrically evaluate the distribution of export sites, er-budding structures were identified as an elevation on the surface of the er with a width of 65-85 nm, extruded from the membrane by at least 50% of their diameter, and covered with a distinctive electron-dense coat ( fig. 1-3, arrowheads). budding sites emanate from three locations in the cell including (a) those associated with the nuclear envelope ( fig. 1) , (b) those associated with the golgi apparatus that are analogous in structure to classical er transitional elements (palade, 1975; sesso et al., 1994) (fig. 2) , and (c) those found in more peripheral regions that lack detectable golgi (fig. 3) . buds found at the tip of tubular projections from the surface of the er had an average diameter of 78 ± 6 nm. although tubular projections were generally shorter than 150 nm (fig. 1 , section 5; arrowheads), they could be as long as 350 nm based on reconstruction from serial-thin sections. buds were covered with an ~8-10-nm-thick electron-dense coat. on grazing sections and at high magnification, the coat of er buds possessed a lattice-like appearance due to a semi-regular array of 4-5-nm elongated particles ( fig. 1, inset) . these coats are similar to those observed in pancreatic acinar cells (merisko et al., 1986) . based on analysis of random sections through ~300 cells (see materials and methods), the average number of buds in a cell was found to be 250 ± 10.45% of budding profiles were found on er tubules located in the vicinity of golgi complexes, whereas 55% were located in regions without noticeable juxtaposition to golgi. 11% of total buds were observed emerging from the nuclear envelope. thus, it is apparent that a substantial level of membrane exiting the er appears to do so from sites distant from golgi elements. the overall average density of total er buds based on the cross-sectional volume of the cytoplasm was found to be 0.5 buds per pom 3, or 0.14 buds per ixm 2 of total er surface. however, the average density of er-budding profiles found within the pericentrosomal area containing the golgi apparatus (referred to as the golgi exclusion zone) was five-to sevenfold higher (3.3 buds per txm 3 of cytoplasm or 0.8 buds per ixm 2 of er surface) than the average value found on the total er membrane. outside of the golgi region, the overall mean density was 1.5-3-fold lower (0.22 buds per ixm 3 cytoplasm or 0.04 buds per ixm 2 of er surface) than the average values found for the cell. the markedly increased budding density around the golgi apparatus is consistent with the highly focused export activity observed from er transitional elements present in the golgi region of pancreatic acinar cells (palade, 1975) . figure 1 . export sites adjacent to the nuclear envelope of rbl cells. six consecutive serial sections through a vtc adjacent to the nuclear envelope. er buds (arrowheads) emerging from the nuclear envelope (section 1) and parallel er membrane (stars in section 2--6) are facing vtcs. (inset) higher magnification view of two er buds. the slice of the section presented in the inset encompasses either only the coat (right), or both the coat and the membrane and the luminal part of a bud (left). individual 4-5-nm electron-dense particles arranged in a semiregular pattern (arrows.) notice the same appearance of the coat under lower magnification of serial sections that contain a honeycombed appearance consisting of a semiregular array of electron-dense particles (arrows). adjacent to buds, we observe a typical pleomorphic element (large asterisk in section 5) within a vtc that has numerous tubular projections (small asterisk) in adjacent thin-sections (small asterisks in sections 4 and 6), indicative of its fenestrated structure. tubules in the fenestrated elements of the vtc possess a dark dense coat (arrows in sections 4 and 6) typical of those found on golgi compartments and are readily distinguishable from the alveolate coat associated with copii buds emanating from the er. bar, 0.1 ixm. figure 2 . export complexes adjacent to the pericentrosomal golgi region of rbl cells. 15 consecutive serial sections through three (a, b, and c) golgi-adjacent export complexes (encircled by dotted lines) are shown with er buds (arrowheads). export complex a contains 13 er-derived buds, export complex b contains 11 buds, and export complex c contains seven buds. note the characteristic cup-shaped appearance of er bud-bearing zones especially evident for export complex b. to develop a detailed understanding of the topological organization of e r export throughout the cytoplasm, we carried out a morphological reconstruction of those sites that were not adjacent to golgi elements, referred to as peripheral sites. a n analysis of peripheral sites allows us to discriminate the structure of er-derived intermediates from that of the fenestrated cis-golgi network (cgn), which is always associated with er buds near the golgi. er buds on peripheral sites (fig. 3) typically emanated from short stretches of er membrane. these regions were separated by long distances from other similar budding foci. frequently, er buds found on different cisternae were closely juxtaposed and faced each other. these features are more evident in fig. 4 (a and b) , which presents an overlapping reconstruction of serial sections of the peripheral site shown in fig. 3 . budding profiles (blue) pro-trude from the er (green) into a central region containing a collection of vesicles and tubular elements comprising vtcs (red). this typical organization of peripheral sites is also characteristic of budding sites associated with the nuclear envelope (fig. 1) and budding sites adjacent to the golgi stack (fig. 2) . the close topological relationship between er buds and distinct vtcs suggests that these structures function as a compact morphological unit that we now refer to in its entirety as an export complex. morphometric analysis of peripheral export complexes revealed that these sites typically contained two to six buds emanating from the er, although this could approach a value of 20 for some exceptional, larger clusters ( fig. 5 a, closed circles) . these sites had an average number of buds per site of 4.4 ___ 0.3 (table ii) , a value that was slightly smaller than the average number of buds per site found in export complexes adjacent to golgi (6.1 +_ 0.4 buds per site; table ii) . by including only er-budding profiles facing vtcs, we were able to estimate a "local" bud density in these export complexes. on average, this value was 17.2 +__ 2.5 buds per i~m 2 of er surface, which, on a relative scale, is 125 times higher than the average bud density found on the total er surface (0.14 buds per ixm 2) and approximately five times higher than the bud density found on the surface of the er within the golgi exclusion zone. the apparent nonrandom distribution of er buds led us to quantitatively estimate the probability of a given bud having proximity to a second bud in the cell. for this purpose, we constructed a series of concentric shells of equal volume that radiate outward from the center of randomly chosen buds. the radius of the first internal shell was arbitrarily assigned a value of 0.1 txm to encompass the entire tip of a budding structure. each increment in the diameter of successive shells extending outward from the first shell progressively decreased in dimension to encompass the same volume in three-dimensional space. given the average number of buds in a cell (~250), the volume of cell cytoplasm (560 i~m3), and the volume of a shell (4.2 × 10 .3 i~m3), if buds assumed a strictly random distribution, then the probability of encountering another bud in a given concentric shell would remain equal with a value of 0.18% ( fig. 6, diamonds) . however, if buds were confined to regional foci, the probability of encountering a second bud would be high in the first series of concentric shells, and then fall off very rapidly with increasing distance in threedimensional space. the results of such an analysis are shown in fig. 6 where we have plotted the probability of encountering a bud relative to its location in sequential concentric shells of equal volume (fig. 6, open circles) , or relative to the diameter of the outermost surface of a given shell in which a bud is found (fig. 6, closed circles) . it is clear that the distribution did not follow that predicted for a strictly random budding event. the probability of detecting a second bud within the first 50 consecutive shells having up to a 0.6-1~m outer shell diameter markedly exceeded that of a random distribution (fig. 6) . after a plateau at a value similar to that calculated for a random distribution (up to an outer diameter of 0.8 ixm or 100 shells), the probability fell dramatically within the three-dimensional space defined by the outermost shell examined (~1.2 txm). we conclude that budding is not a random event along the surface of the er, but rather is remarkably restricted to regional hot spots of budding activity associated with export complexes. as illustrated in fig. 4 , er-budding profles predomi-nately faced into an area filled with vtcs in vivo. vtcs, defined as a group of four or more 60-80-nm vesicular profiles with a characteristic shape and coat, were never detected in regions lacking adjacent er buds. the membrane profiles of vtcs were distinct from the er and found to be more variable in size compared to the rather homogeneous appearance of buds emanating from the er surface. the average diameter of vtcs based on serialthin sections was found to be ~0.4 cm (table ii) with a range that varied from ~0.2 ~m to >1 ~m (fig. 5 b) . although technical limitations do not allow us to make a complete three-dimensional reconstruction of membrane continuities between elements of vtcs, individual profiles often appeared to be continuous in several consecutive sections and consisted of short tubules that could be connected together in a more fenestrated structure (fig. 1, asterisks) . tubules within vtcs characteristically had a dense copi-like coat at their tips (figs. 1 and 3, arrows) (melancon et al., 1987; orci et al., 1993a) . the presence of a copi-like coat within vtcs is consistent with the numerous morphological studies that have demonstrated that these pre-golgi intermediates are a major site of copi localization when examined using indirect immunofluorescence (lippincott-schwartz, 1993; aridor et al., 1995) or tem (oprins et al., 1993; pind et al., 1994a; griffiths et al., 1995b) . to estimate the total number of elements within a vtc, we assumed that each profile appearing in an individual section was, in fact, a separate vesicle or tubule. from this assumption, the average number of elements was determined to be 32 +__ 4 (table ii) . clusters generally contained between 10 and 60 vesicular-tubular elements, although some of the clusters contained as many as 150 elements (fig. 5 c) . although these values are undoubtedly an overestimate of the total number of luminally distinct elements, given that tubules within vtcs are likely to extend across several consecutive thin-sections, they serve as a useful numerical approximation for the relative size and composition of vtcs. when we compared the number of elements found in peripheral vs golgi-adjacent vtcs, we observed very similar values, suggesting they are of comparable size (fig. 5 c) . whereas peripheral vtcs were generally spherical in shape, those adjacent to golgi possessed a semispherical shape with the cgn occupying one hemisphere, and the other hemispheres facing er membranes. using the disector method to estimate the total number of vtcs per cell (sterio, 1984 ) (see materials and methods), we found an average value of 74 _ 11 clusters per cell. this level is similar to that observed using more indirect tem methods (buccione et al., 1996) or indirect immunofluorescence in other cell lines (plutner et al., 1992; aridor et al., 1995) . to correlate our morphological observations with previous in vitro biochemical studies, we used semi-intact nrk cells to examine the potential role of copii components in the formation of er-derived buds and vtcs (plutner et al., 1992; peter et al., 1993; balch et al., 1994; pind et al., 1994b) . semi-intact cells incubated for 30 min at 32°c in figure 4 . three-dimensional reconstruction of a peripheral export complex. membrane contours shown in fig. 3 are illustrated at a magnification of 160,000. transparencies containing the membrane contours were scanned, overlayed, and coaligned based on the position of mitochrondria and er. green denotes er cisternae, blue denotes er buds, and red denotes tubules and vesicles of vtcs. vesicular membrane contours within the vtc whose luminal continuity to the surrounding er membranes was evident in consecutive sections were denoted in green. more intense shades of the same color reflect distance from the uppermost section. the alveolate coats of er buds, where evident, are dictated by stipples. images are presented either individually with the section number indicated (bottom two rows), or as an overlay containing four images (left) for clarity or all eight images (right). the resulting reconstruction shows the clustered structure of a typical vtc, surrounded by er-bearing buds occasionally penetrating the periphery of a vtc, as evidenced by the coated portions of er tubules (blue) adjacent to vtc tubules (red). the absence of cytosol failed to generate detectable vtcs (not shown). this result is consistent with the fact that preexisting vtcs are unstable during permeabilization (aridot et al., 1995) and that cytosol contains essential soluble components of the copii machinery required for the export of cargo from the e r (barlowe et al., 1994; kuge et al., 1994) . vtcs generated in vitro in the presence of cytosol are nearly identical to those observed in vivo. we have previously shown that they are composed of a compact network of tubules and vesicles that lack direct luminal connections to e r membranes and frequently label positively for b-cop using immunoelectron microscopy pind et al., 1994a) . however, er-budding profiles, like those observed adjacent to vtcs in vivo (figs. 1-3, arrowheads), were rarely observed in vitro. since previous studies used mild fixation conditions in conjunction with an immunodiffusion procedure to label vsv-g in vtcs , we reasoned that er buds may be labile structures. we therefore applied more stringent fixation and embedding conditions to preserve ultrastrucrural details (see materials and methods). under these conditions, er buds were observed that had a characteristic coat resembling those found in vivo and were only detected adjacent to vtcs (not shown), suggesting that even in semi-intact ceils, budding is restricted to specialized regions of the er, to examine the formation of buds and their relationship to vtcs in vitro, we made use of the nonhydrolyzable analog of gtp, gtp~/s, which permanently activates sarl and other gtpases, leading to stable coat assembly and accumulation of buds. interestingly, we observed for the first time using strong fixation conditions that nascent budding profiles generated in the presence of gtp~/s had not only a cluster appearance (pind et al., 1994a) , but also frequently had a distinctive "beaded necklace" appearance with each vesicle being a bead (fig. 7, b and c) . the vesicles were nearly identical in size but lacked luminal continuity. these strings of vesicles were similar to the shorter necklaces sometimes observed in vivo under normal incubation conditions (fig. 7 a) . these necklaces were confined to only a small fraction of the total er surface. by analyzing sections through >600 individual necklaces, we have never detected them to have more than one connection to the er membrane, supporting the possibility that each string grows from a local area of budding activity. these observations suggest that budding continues outer diameter of shell (lid) figure 6 . probability of a given bud having proximity to a second bud in the cell. randomly chosen er buds present in rbl cells were assigned as the center of reference, and distances between it and any other buds present in 30 consecutive serial sections were determined by building a series of concentric shells with a constant volume of 0.0042 ixm 3, corresponding to the volume of the first internal shell having a diameter 0.2 ixm (to encompass a single bud) as described in the materials and methods. the probability was determined by counting the number of buds detected in each shell relative to the total number of buds detected (percentage of total). this value is plotted as shell number in which a second bud was found (open circles) or relative to the outermost diameter of a particular shell (closed circles). the calculated probability of a second bud having a completely random distribution in the cell (0.18 %) is presented for comparison (diamonds). in the absence of gtp hydrolysis, but that the vesicles fail to complete separation from one another. upon immunolabeling, we found accumulated vesicles to be substantially enriched in vsv-g (pind et al., 1994a) and components of both copi (13-cop) (pind et al., 1994a; griffiths et al., 1995b) and copii coats (sec13 and sec 23) (fig. 8, a-c) . to establish that necklaces formed in response to a specific block in copii coat disassembly, we examined the ef-fect of an activated, gtp-restricted mutant of sarl, sarl[h79g], which promotes vesicle accumulation in vivo and in vitro (aridor et al., 1995; kuge et al., 1994) . examination using strong fixation conditions revealed a striking similarity to gtp~/s-formed structures. vesicles were detected as both clusters (fig. 7 d) or as necklaces (fig. 8 e) emerging from restricted regions of the er. as expected, coats were enriched in the copii components secl3p (arrowheads in a, c, and d) and sec23p (arrowheads in b, and e) as shown by the distribution of gold particles using specific antibodies. note that the cisternal portion of the golgi apparatus with the trans face labeled in c and f remains unlabeled by sec13p-and sec23p-specific antibodies, whereas clusters (arrows) that are closely adjacent to the golgi complex contain both secl3p and sec23p (c and f). bar, 0.05 txm. bannykh et al. organization of er export complexes (fig. 8 e) and sec23p (fig. 8 f) . no copi staining could be detected using an antibody specific for 13-cop in these structures (not shown) (aridor et al., 1995) . to examine the role of copii in vsv-g concentration and the appearance of vtcs in export complexes, we used semi-intact cells infected with a temperature-sensitive form of vsv-g whose transport is blocked at the restrictive temperature (39.5°c) (lafay, 1974; plutner et al., 1992) . transfer of cells to the permissive temperature (32°c) results in the migration of a synchronous wave of ts045 vsv-g from the er to vtcs and subsequent golgi compartments (plutner et al., 1992; balch et al., 1994) . after incubation at 32°c in vitro, cells were fixed and stained with an antibody specific for the cytoplasmic tail of vsv-g using the immunodiffusion protocol . as shown in table iii , incubation in the presence of wildtype sarl had little effect on er export as judged by the abundance of vtcs detectable in vitro. in contrast, the guanosine diphosphate (gdp)-restricted form of sarl (sarl [t39n] ) drastically reduced the formation of vesicles and vtcs (table iii) , demonstrating the essential need to activate sarl to promote membrane flow from the er through export complexes. in contrast, incubation in the presence of the gtp-restricted mutant (sarl[h79g]) caused a dramatic accumulation of vesicles in clusters and an approximate twofold increase in the apparent number of clusters per section that could be detected (table iii) . when we determined the density of vsv-g in clusters formed in the presence of the sarl-gtp restricted mutant, it was approximately five-to sixfold higher than that found in the er before incubation (table iii) . this fold-concentration was identical to that observed in vtcs present in control incubations lacking inhibitors (table iii) . the five-to sixfold increase in the density of vsv-g in vesicles that accumulate in the presence of the mutant demonstrates that vsv-g is concentrated during packaging into copii-coated vesicles. to develop a three-dimensional view of export complexes, we applied for the first time a modification of the quickfreeze, deep-etch methodologies used previously to visualize vesicles budding from the plasma membrane (heuser, 1980) and golgi compartments (weidman et al., 1993) . the approach is particularly applicable to semi-intact cells where the cytosol can be readily washed away to reveal structural features of the er membrane surface. after incubation in vitro in the presence of atp and cytosol, semi-intact cells were fixed, rapidly frozen, and fractured to expose internal membranes. after etching and replication, intracellular organelles are rendered visible as three-dimensional structures. the surface of the er was readily distinguishable from other subcellular compartments by the presence of ribosomes or, in the case of the nuclear envelope, additionally, nuclear pores. adjacent to the surface of the er, we frequently observed compact structures of similar size and apparent vesicular-tubular composition to vtcs observed in thin-sections ( fig. 9 a) . these structures were completely absent in incubations that lacked cytosol or atp. they varied in diameter, but were generally ~0.3-0.5 ~m across and ranged from a circular to a more oblong shape under normal incubation conditions. assuming that the distinctive ~80-nm surface undulations correspond to vesicle profiles ( fig. 9 a) and that a cluster can be represented as a sphere with a similar range of diameters, we estimate that vtcs detected in replicas could contain 50-110 individual elements. this value is compatible with the number of vesicular profiles determined by reconstruction from serial thin-sections. to identify whether the above structures formed in the presence of cytosol and atp contained er-derived cargo proteins such as vsv-g, nrk cells were infected with ts045 vsv at the restrictive temperature. after permeabilization and incubation in vitro at 32°c, cells were immunolabeled for vsv-g using the immunodiffusion protocol and replicas were prepared. while a combination of prolonged incubation using mild fixation conditions to label vsv-g and the presence of prominent "gold shadows" from the etching and replication reduces the ability of the technique to reveal surface features of these immunogold-labeled clusters, the distribution of vsv-g reveals the striking role of copii in concentrative export. before incubation of semi-intact cells at 32°c, gold detected on the surface of the er (fig. 10 a, arrowheads) and nuclear envelope (fig. 10 b, arrowheads) was distributed in an apparent random manner throughout the er cisternal network (plutner et al., 1992; balch et al., 1994) . the surface density of vsv-g in these er membranes was 32 +-6 gold particles per p~m 2. in contrast, incubation for 45 min in the presence of cytosol and atp led to a dramatic change in the distribution of vsv-g, rearranging gold particles to a limited number of vtcs that were well isolated from each other (fig. 10 c) . the density of gold over vtcs projected as a flat surface parallel to the er membrane (referred to as a planar projection) was ~850 -+ 250 gold particles per p~m 2, a value markedly higher than that observed in the plane of the er membrane before incubation. we next used replicas to follow the effects of gtp'ys and the sarl-gtp restricted mutant on budding and concentration of vsv-g. as shown in fig. 10 (b and c) , incubation of noninfected semi-intact cells in the presence of gtp~/s led to the accumulation of vesicles ~8 0 nm in diameter that, consistent with t e m (fig. 7 , b and c), had a zig-zag necklace-like appearance when collapsed on the surface of the er. immunolabeling of replicas formed in the presence of gtp~/s revealed that vsv-g reached a density of 800 _ 200 gold particles per txm ~ in planar projection within clusters. when incubations were carried out in the presence of the sarl-gtp restricted mutant, necklaces (not shown) and compact vesicular clusters were also observed on replicas (fig. 9 d) . in the presence of the sarl mutant, vsv-g reached a density of 700 _+ 200 gold particles per ~m 2 in a planar projection of the cluster (fig. 10 e) , reinforcing our previous observations that the budding activity associated with export complexes involves concentration. we have provided the first quantitative, stereological de-scription of the three-dimensional organization of cellular structures involved in transport of cargo from the e r to the golgi apparatus. export complexes have a hierarchial organization that can be conceptually divided into three tiers (fig. 11 a) . the first tier (fig. 11 a, dotted box) consists of closely adjacent buds on a single e r cisterna. each can give rise to an individual string or group of erderived buds containing copii coats. these budding loci were limited to specific regions of the e r in vivo, suggesting the existence of a defined number of export sites in the living cell. the second tier (fig. 11 a, cylindrical region outlined by dashed lines) comes from the observation that buds on one cisterna were often found in close proximity to budding profiles emanating from e r cisternae derived from distantly connected regions of the er. the third tier of organization encompassing the entire export complex ( fig. 11 a, solid box) includes er-derived buds that face into a region housing a central vtc. tubular elements within vtcs contain distinctive copi coats and are luminally discontinuous with the er. while professional secretory figure 10 . vsv-g is concentrated in er-derived vesicles and vtcs. nrk cells infected with ts045 vsv at the restrictive temperature (39.5°c) to retain vsv-g in the er were permeabilized with digitonin (plutner et al., 1992) and either fixed immediately (a and b) or incubated for 45 rain at the permissive temperature (32°c) in the presence of cytosol and atp (c), or additionally supplemented with 1 ixm sarl[h79g] (e). after incubation, cells were fixed and vsv-g labeled with 10 nm (a-c, and e) or 6 nm (d) gold particles using the immunodiffusion protocol as described in the materials and methods. cells were either prepared for thin-section tem (d) or for quickfreeze, deep-etch replication (a-c, and e). at the restrictive temperature, vsv-g was uniformly distributed within the er membrane (a) or the nuclear envelope (b). after a shift to the permissive temperature (b-e), vsv-g was concentrated in newly formed 80-nm vesicles associated with export complexes. (arrowheads) location of 6 nm (d) or 10 nm (a-c, and e) gold particles corresponding to the distribution of vsv-g. due to the use of an immunodiffusion protocol before preparation of replicas and the high density of label, extended shadows from the gold particles partially obscure membrane outlines. bar, 0.1 i~m. cells such as those found in the pancreas confine export predominately to a single transitional region juxtaposed to the cis face of the golgi apparatus (palade, 1975) , the two different cell lines used in the present study were found to have export complexes distributed throughout the cytoplasm. our studies provide evidence that budding from the e r occurs in areas of intense morphological specialization. each level of organization is discussed in detail below. while there is an apparent random distribution of export complexes in the cytoplasm, we found a very high degree of organization in the distribution of e r buds in the cell. we observed not only a high local density on the same stretch of e r membrane (fig. 11 , tier i, dotted boxes), but found distantly connected e r bud-bearing zones encircling the same vtc (fig. 11 , tier ii, dashed cylindrical region), suggestive of a regional specialization of the copii export machinery. consistent with this interpretation, buds and vesicles accumulated in semi-intact cells in the pres-ence of gtp~/s or the sar1[h79g] mutant strongly labeled with antibodies specific for mammalian homologues of the yeast components secl3p and sec23p. both the gtp-ys and the gtp-restricted sarl[h79g] mutant led to the formation in vitro of er-derived vesicle clusters and strings of vesicles with a necklace-like appearance. vesicles accumulated as necklaces had no obvious continuity between the lumen of individual vesicles, suggesting that membrane fission had gone to completion. moreover, in both thin-section and replicas, clusters that formed in the presence of gtp'ys or the sarl gtprestricted mutant clearly had a more vesicular surface appearance than those formed in the absence of inhibitors. the fact that separation of vesicles appears to be blocked in the absence of gtp hydrolysis raises the distinct possibly that the function of sarl is normally required for this event. given the fact that uncoating occurs rapidly after budding (aridor et al., 1995) , coat disassembly could be associated with release of vesicles from necklaces. the morphological effect of gtp~s on the budding from the e r is very different from its effect on golgi membranes incubated under identical conditions. in the latter case, buds appearing in replicas are isolated, single structures and are uniformly distributed throughout the entire golgi surface (weidman et al., 1993 ). the regional confinement of budding in e r membranes to necklaces or clusters therefore also supports our interpretation that export occurs from areas of luminal and/or membrane specialization. since budding from the e r frequently occurs from the tips of short, coated tubules emanating from e r cisternae, clusters and necklaces could be derived from either sequential or synchronous fission of these tubular elements. in contrast with the fact that export complexes observed in vivo appear to be completely surrounded by budding profiles from topologically distant e r cisternae (fig. 11 a, tier ii), semi-intact cells lacked this feature (fig. 11 b) . thus, permeabilization destroys confinement of several sites to one area and allows them to form in a more random fashion. we have previously noted that er-derived vesicles and downstream compartments are not released from semi-intact cells during incubation in vitro at 32°c (beckers et al., 1987) . in contrast, assays that reconstitute vesicle budding from semi-intact yeast cells (baker et al., 1988) release free 60-nm copii-coated vesicular carriers (barlowe et al., 1994) . the inability of mammalian semiintact cells to release vesicles suggests that vesicles are tethered to a scaffold of unknown composition. in yeast, either vesicles are not linked to such a scaffold, or this aspect has not been successfully reconstituted. in either event, while the striking degree of morphological specialization observed in mammalian cells may contribute to the overall efficiency of budding and transport in the early secretory pathway, it is apparently not essential. we have previously suggested that export from the e r is accompanied by concentration of vsv-g . this point was the subject of a recent debate (balch and farquhar, 1995; griffiths et al., 1995a) . we have now applied an independent approach using quick-freeze, deepetch methodologies in conjunction with immunolabeling of vsv-g to generate three-dimensional replicas that allow us to directly assess the concentration of vsv-g in er-derived vesicles. we found, on average, a value of ~800 gold particles per txm 2 in planar projection of clusters accumulated in either the absence or presence of gtp~s, or in the presence of the activated sarl-gtp restricted mutant. the inclusion of the inhibitors prevents further rounds of vesicle budding, ensuring that we are examining concentration associated with export from the er. a planar projection, however, is not a good approximation of the total surface area available on clusters for antibody binding. since only the external surface of vesicles found on the perimeter of clusters is available for antibody binding pind et al., 1994a ) (see fig. 11 d) , a more reasonable estimate of vsv-g density can be determined if we assume that the antibody has access to the outer-half of a shell of 80-nm vesicles that occupy the perimeter of a 0.4-~m sphere (the size of a typical vtc). compared with the surface area of the planar projection (~0.13 ~m2), the surface area of such a population of vesicles corresponds to a value of ~0.7 txm 2. this is an increase of approximately fivefold over the planar projection. therefore the actual surface density of vsv-g in clusters observed in replicas is 800 gold particles per i~m 2 divided by 5 or 160 gold particles per ixm 2. this value, when compared to the average surface density of vsv-g before incubation in vitro (32 gold particles per ~m2), sugfigure 11 . diagram summarizing the three tiers of organization of er export complexes in vivo (a) and in vitro (b). (a) an individual er cisterna contains a collection of closely opposed buds that define a local transitional region (light zone with buds on the er). this specialized region is dominated by the presence of copii coats and is referred to as tier 1 (box outlined with dotted line). tier ii (cylindrical region outlined by dashed lines) includes buds on distantly connected er strands that face a central vtc consisting of a collection of distinct vesicular-tubular elements that have copi coats. tier iii includes the entire export complex and is outlined by the box with the solid line that encompasses both er buds and a central vtc. the possible elevated concentration of copii and copi coat components within the local cytoplasm of export complexes is depicted by small dots and lines. (b) in semi-intact cells, there appears to be a more limited number of er buds associated with the local transitional region defined by tier i (box outlined with dotted lines). the tier ii level of organization is completely missing in semi-intact cells, as the association of distantly connected er strands appears to be lost during cell permeabilization. however, tier iii (box outlined with solid line) is maintained, highlighting the juxtaposition of vtcs to buds on one er strand. gests that vsv-g is concentrated five-to sixfold during budding from the er. the fold-concentration detected here is very consistent with that observed previously using serial thin-sections pind et al., 1994a) or in the present studies in the presence of the sarl-gtp restricted mutant (table iii) . these results confirm the validity of our previous technical advancements balch and farquhar, 1995) and now firmly establish that cargo is concentrated during er export . sequential coupling between copii and copi vesicle coats in endoplasmic reticulum to golgi transport estimation of surface area from vertical sections reconstitution of sec gene product-dependent intercompartmental protein transport beyond bulk flow atp-coupled transport of vesicular stomatitis virus g protein between the endoplasmie reticulum and the golgi vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum copii: a membrane coat that forms endoplasmic reticulumderived vesicles copii: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the golgi complex regulation of constitutive exocytic transport by membrane receptors: a biochemical and morphological study localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments transport into and out of the golgi complex studied by transfecting cells with cdnas encoding horseradish peroxidase differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and trans-golgi network density of newly synthesized plasma membrane proteins in intracellular membranes. i. stereological studies the dynamic nature of the golgi complex the bulk-flow hypothesis: not quite the end immunocytochemical localization of [3-cop to the er-golgi boundary and the tgn disruption of endoplasmic reticulum to golgi transport leads to the accumulation of large aggregates containing 13-cop in pancreatic acinar cells three-dimensional visualization of coated vesicle formation in fibroblasts microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step sarl promotes vesicle budding from the endoplasmic reticulum but not golgi compartments envelope viruses of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups iii and v coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum bi-directional membrane traffic between the endoplasmic reticulum and golgi apparatus kinesin is the motor for microtubule-mediated golgi-to-er membrane traffic immunocytochemical analysis of the transfer of vesicular stomatitis virus g glycoprotein from the intermediate compartment to the golgi complex mitotic golgi fragments in hela cells and their role in the reassembly pathway the gotgi complex: in vitro veritas? celt the reorganization of the golgi complex in anoxic pancreatic acinar ceils mammalian sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm coated vesicle assembly in the golgi requires only coatomer and arf proteins from the cytosol bfa bodies: a subcompartment of the endoplasmic reticulum lntracellular aspects of the process of protein transport. science (wash. dc) evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment 13-cop is essential for biosynthetic membrane transport from the endoplasmic reticulum to the golgi complex in vivo 13-cop is essential for transport of protein from the endoplasmic reticulum to the golgi in vitro rabl and ca 2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to golgi transport participation of the endoplasmic reticulum chaperone calnexin (p88, ip90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator morphological analysis of protein transport from the endoplasmic reticulum to golgi membranes in digitonin-permeabilized cells: role of the p58 containing compartment biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the ceil surface distribution of the intermediate elements operating in er to golgi transport identification, by a monoclonal antibody, of a 53-kd protein associated with a tubulo-vesicular compartment at the cis-side of the golgi apparatus identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to golgi apparatus a threedimensional reconstruction study of the rough er-golgi interface in serial thin sections of the pancreatic acinar cell of the rat human sec13rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum estimating number, mean sizes and variations in size of particles in 3-d specimens using disectors anterograde and retrograde traffic between the rough endoplasmic reticulum and the golgi complex segregation of ergic53 and the mammalian kdel receptor upon exit from the 15°c compartment replication of coronavirus mhv-a59 in sac cells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e1 glycoprotein of mouse hepatitis virus-a59 stereoiogical methods. 1. practical methods for biological morphometry a principle for counting tissue structures on random sections integrated stereological and biochemical studies on hepatocytic membranes. ii. correction of section thickness effect on volume and surface density estimates golgi membrane dynamics imaged by freeze-etch electron microscopy: views of different membrane coatings involved in tubulation versus vesiculation speculations based on the morphology of the golgi system in several types of protein secreting cells we thank dr. g. palade, dr. m.g. farquhar, and michael mccaffery for their many helpful comments concerning the em. this work was supported by grants from the national institutes of health (gm 42336; ca 586689) (to w.e. balch), and postdoctoral fellowships from the human frontier science program organization, muscular dystrophy association (to t. rowe), and the cystic fibrosis foundation (to s. bannykh). this study made extensive use of core b (immunoelectron microscopy) in ca 586689.received for publication 22 april 1996 and in revised form 11 june 1996. a third tier of organization of export sites was found in the striking relationship between flanking er-connected budding profiles and vtcs to form the functional morphological unit we refer to as export complexes (fig. 11 a, area enclosed by box with solid line). images reconstructed from conventional tem and those observed in replicas yielded identical results. the distinctive morphological characteristics of the export complexes reconstructed in the present studies are consistent with previous qualitative morphological descriptions (saraste and kuismanen, 1984; schweizer et al., 1988 schweizer et al., , 1990 saraste and svensson, 1991) and a recent study in which a hrp-tagged reporter protein was used to characterize the organization of the er/golgi region using immunocytochemistry (stinchcombe et al., 1995) . the replicas were particularly striking in that they allowed us to visualize for first time the overall compact composition of vtcs and their localization to specific foci found on the er surface.the overall topological organization of export complexes fits well with the proposed function of the er in the sorting and concentration of cargo during budding via copii coats, and the subsequent coupled recycling of proteins from vtcs via copi coats (aridor et al., 1995) . the close association of these two sorting stations (er and vtcs) may provide for increased efficiency in er to golgi transport and/or promote more rapid exchange of coats within the confines of the complex where a higher local coat concentration may be found. consistent with this proposal, incubation of pancreatic acinar cells in the absence of atp (merisko et al., 1986) , 1992) , is also considered to be a site of membrane recycling. it may be an enlarged variation of the more compact vtcs, reflecting the intensity of vesicular traffic in this region of the cell. in general, our studies have provided insight into the fundamental morphological organization of the first steps in the secretory pathway that promote the movement of cargo from the er to the golgi complex. there has been considerable controversy regarding the morphological organization of this stage of secretory pathway, given the complexity of the pre-golgi region and the frequent use of reduced temperature to augment the visibility of intermediates (vtcs). our ability to provide a description of the three-dimensional organization of export complexes at sites distant from the golgi apparatus under normal incubation conditions now illustrates their basic organization in living cells and their essential role in er to golgi transport. key: cord-351881-qea4b0i5 authors: eck, melanie; durán, margarita garcía; ricklin, meret e.; locher, samira; sarraseca, javier; rodríguez, maría josé; mccullough, kenneth c.; summerfield, artur; zimmer, gert; ruggli, nicolas title: virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs date: 2016-02-19 journal: vet res doi: 10.1186/s13567-016-0318-0 sha: doc_id: 351881 cord_uid: qea4b0i5 porcine reproductive and respiratory syndrome virus (prrsv) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. the vaccines currently available on the market elicit only limited protection. recombinant vesicular stomatitis virus (vsv) replicon particles (vrp) have been used successfully to induce protection against influenza a virus (iav) in chickens and bluetongue virus in sheep. in this study, vsv vrp expressing the prrsv envelope proteins gp5, m, gp4, gp3, gp2 and the nucleocapsid protein n, individually or in combination, were generated and evaluated as a potential vector vaccine against prrsv infection. high level expression of the recombinant prrsv proteins was demonstrated in cell culture. however, none of the prrsv antigens expressed from vrp, with the exception of the n protein, did induce any detectable antibody response in pigs before challenge infection with prrsv. after challenge however, the antibody responses against gp5, gp4 and gp3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control vrp. no reduction of viremia was observed in the vaccinated group compared with the control group. when pigs were co-vaccinated with vrp expressing iav antigens and vrp expressing prrsv glycoproteins, only antibody responses to the iav antigens were detectable. these data show that the vsv replicon vector can induce immune responses to heterologous proteins in pigs, but that the prrsv envelope proteins expressed from vsv vrp are poorly immunogenic. nevertheless, they prime the immune system for significantly earlier b-cell responses following prrsv challenge infection. infections with porcine reproductive and respiratory syndrome virus (prrsv) causes reproductive failures in sows [1] and respiratory disorders particularly in young pigs [2] , which results in important economic losses worldwide [3, 4] . recently, highly pathogenic prrsv strains have emerged in china [5] and eastern europe [6] . prrsv is an enveloped positive sense singlestranded rna virus belonging to the family arteriviridae within the order nidovirales [7] . two prrsv genotypes can be distinguished, type 1 prrsv of european origin and type 2 prrsv originating from north america and china, both spreading worldwide with high genetic and antigenic diversity [8, 9] . the prrsv genome consists of at least 10 open reading frames (orf). orf 1a and 1b encode the non-structural proteins from two polyproteins pp1a and pp1ab that are further processed proteolytically, as well as two proteins nsp2tf and nsp2n resulting from ribosomal frameshifts within the nsp2 gene (for a detailed review see [10] ). the remaining orfs encode the structural proteins on subgenomic messenger rnas. orf 2a, 2b and 3-7, encode the glycoprotein 2 (gp2) also termed gp2a, the non-glycosylated protein 2b also termed e, the glycoproteins gp3, gp4, gp5, the non-glycosylated membrane protein m (from orf6) and open access *correspondence: nicolas.ruggli@ivi.admin.ch 1 institute of virology and immunology ivi, sensemattstrasse 293, 3147 mittelhäusern, switzerland full list of author information is available at the end of the article the nucleocapsid protein n (from orf7), respectively (reviewed in [11] ). recently, an alternative orf5a protein was identified as a minor component of the equine arteritis virus (eav) [12] and the prrsv virions [13] . gp5 and m form a disulphide-linked heterodimer that is essential for the formation of infectious particles [14, 15] . for eav, the glycoproteins gp2, gp3 and gp4 form a heterotrimeric complex that is stabilised by disulphide bonds, which has not been demonstrated for prrsv yet (reviewed in [11] ). the prrsv gp5-m and gp2-gp3-gp4 complexes are linked essentially through non-covalent interactions between gp5 and gp4 [16] . the basic protein n associates with the viral rna genome to form the nucleocapsid. n is the most immunogenic prrsv structural protein. it elicits a strong antibody response a few days post infection (pi). these antibodies do however not neutralize the virus and are therefore not protective [17] . the major neutralizing epitopes are found on gp5 [18] [19] [20] [21] and gp4 [22] [23] [24] which are also the most diverse structural proteins between isolates [25] . neutralizing epitopes were also found on m, gp3 and gp2 [26] [27] [28] , but their contribution to protection is unclear. gp5 co-expressed with m elicits a better neutralizing ab response than gp5 alone [29, 30] . however, neutralizing antibodies appear typically several weeks only after the onset of the first antibody response, simultaneously with clearance of the virus from the bloodstream [21, 31] . the development of vaccines against prrsv has been only partially successful so far and remains a challenging task (for comprehensive reviews, see [32] [33] [34] ). there are currently two types of prrsv vaccines on the market: modified live-virus vaccines (mlv) and inactivated vaccines [35] [36] [37] . mlv are typically more efficacious than inactivated vaccines [38, 39] . numerous alternative prrsv vaccine approaches have been explored with limited success so far (reviewed in [32, 40] ). these efforts include for instance dna vaccines, subunit and peptide vaccines, viral vector vaccines and plant-derived vaccines [30, [41] [42] [43] [44] [45] [46] . propagation-incompetent recombinant vesicular stomatitis virus (vsv) represents yet another vector vaccine approach. recombinant vsv replicons lacking the vsv glycoprotein (g) gene and carrying genes of interest instead can be packaged in virus replicon particles (vrp) with high infectious titres using a complementing g-expressing cell line [47] . such vrp were used successfully in the past to induce protection against sars coronavirus in a mouse model [48] , influenza a virus (iav) in chickens and mice [49] [50] [51] , and bluetongue virus (btv) in sheep [52] . vsv vrp are safe due to the lack of glycoprotein g expression, preventing assembly and spread of virus particles. pigs have typically no pre-existing immunity against vsv. thus, vsv replicons represent an attractive novel vaccine platform for pigs. they have however not been evaluated in pigs yet. in the present study, vsv vrp were generated to express different combinations of the major and minor prrsv glycoproteins. the immunogenicity and protective potential of these vrp were assessed in pigs. marc-145 cells (atcc, lgc standards) were grown in dulbecco's modified eagle medium (dmem; life technologies) supplemented with 10% foetal bovine serum (fbs; biowest). bhk-21 cells were obtained from the german cell culture collection (dszm) and grown in earle's minimal essential medium eagle (mem; life technologies) supplemented with 5% fbs. bhk-g43, a transgenic bhk-21 cell clone expressing the vsv g protein in a regulated manner, were maintained as described previously [47] . the type 1 prrsv strain olot/91 was kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). this virus was a marc-145-adapted variant of the original olot/91 virus and was therefore propagated and titrated in marc-145 cells. for the detection of prrsv proteins, monoclonal antibody (mab) 13e2 directed against prrsv n, and mab vii2d directed against amino acids 73-84 of prrsv gp3 were kindly provided by hans nauwynck (university ghent, belgium). a polyclonal rabbit serum against prrsv gp4 was obtained from luis enjuanes (centro nacional de biotecnología, madrid, spain). this serum was generated at biogenes (germany) with purified recombinant gp4 from prrsv olot/91 expressed with the baculovirus system. the mab 11e10c7 directed against prrsv m, and the mab 3ah9 against prrsv gp5 were a gift from ingenasa (madrid, spain). the rabbit anti-myc antiserum c3956, the mouse anti-flag m2 antibody and the anti-α-tubulin mab were purchased from sigma-aldrich. the mouse anti-ha antibody 12ca5 was from roche. the secondary antibodies goat anti-mouse igg alexa 546 and goat anti-rabbit igg alexa 546 were from molecular probes. the anti-swine igg antibody conjugated with rhodamine was purchased from rockland. the polyclonal rabbit anti-mouse igg coupled with horseradish peroxidase was from dako. the cdna of gp5 and m and the codon-optimized cdna of gp2, gp3 and gp4 from the prrsv olot/91 strain (genbank accession number kc862570) were derived from psl-orf5-orf6 and psl-gp3-2-4, respectively, kindly provided by luis enjuanes (centro nacional de biotecnología, madrid, spain). the codonoptimized cdna of n from the olot/91 strain (genbank accession number agw23409) was synthesized by genscript (piscataway). for generation of recombinant vsv replicons, gp5 or n were inserted into the plasmid pvsv* using mlui and bsteii restriction sites upstream and downstream of the fourth transcription unit, replacing the g gene of vsv in analogy to a previous report [49] . this replicon contained an additional transcription unit at position 5, expressing the green fluorescent protein (gfp) referred to by an asterisk (*) in the vector nomenclature. the resulting plasmids were designated pvsv*δg(gp5) and pvsv*δg(n), respectively. for generation of a dual antigen expression vector, the m cdna was inserted into pvsv*δg(gp5) using xhoi and nhei restriction sites, thereby replacing the gfp gene in the fifth transcription unit. the resulting plasmid was designated pvsvδg(gp5/m). for generation of a triple antigen expression vector, the cdna of gp4, gp3 and gp2 was inserted into a vsvδg vector containing 7 transcription units described recently [53] using the mlui (in case of gp4), xhoi (in case of gp3) and nhei (in case of gp2) restriction sites. the resulting plasmid was designated pvsvδg(gp4/gp3/gp2). for expression of a modified m and gp3 containing a short peptide epitope at the c terminus, the m and gp3 gene, respectively, were inserted without stop codon into the pcmv-3tag-3a plasmid vector (agilent technologies) upstream and in frame with a triple flag epitope (dykddddk)coding region followed by a stop codon. the m-flag and gp3-flag open reading frames were amplified by pcr and inserted into the fourth transcription unit of pvsv*δg, resulting in the plasmids pvsv*δg(m-flag) and pvsv*δg(gp3-flag), respectively. the antigens gp5 and gp4 were modified by fusing a short ha epitope (ypydvpdya) to the c terminus, while gp2 was modified at the c terminus with a short myc epitope (eqkliseedl). the orfs were inserted into the fourth transcription unit of pvsv*δg resulting in the plasmids pvsv*δg(gp5-ha), pvsv*δg(gp4-ha), and pvsv*δg(gp2-myc), respectively. for expression of the gp3 ectodomain (gp3ecto) without the c-terminal transmembrane domain, a gene cassette encoding the amino acids 29-176 of gp3 (numbering according to genbank accession number agw23404) was inserted in frame with the igκ leader sequence and an optimal signalase cleavage site in the mammalian expression vector psectag-2a (invitrogen). in this way gp3ecto was fused to a myc peptide epitope and a histidine tag (6xhis) at the c terminus, followed by a stop codon. this igκ-gp3ecto-myc-6xhis construct was then amplified by pcr and inserted into the fourth transcription unit of pvsv*δg, resulting in the plasmid pvsv*δg(gp3ecto-myc). for expression of iav proteins, the cdna encoding ha, na, np and m2 of iav a/swine/belzig/2/01 (h1n1) were kindly provided by jürgen and olga stech (friedrich-loeffler-institut, greifswald-insel riems, germany). the genes were inserted into the plasmid pvsv* using the mlui and bsteii restriction sites, replacing the g gene of vsv as described [49] . all nucleotide sequences were confirmed by sanger sequencing. the recombinant replicons were propagated in the bhk-g43 helper cell line providing the vsv g protein in trans, yielding infectious vrp with titres of 10 7 -10 8 infectious units (iu)/ ml as described previously [54] . for the titrations, gfp expression was used as readout. for the detection of vrp that did not express gfp, infected cells were fixed with pbs containing 3% paraformaldehyde (pfa) for 20 min, washed with pbs containing 0.1 m (w/v) glycine and then permeabilized with 0.25% (v/v) triton x-100. the cells were incubated with a rabbit anti-vsv serum and subsequently with a goat anti-rabbit horseradish peroxidase conjugate (dako) and stained with 3-amino-9-ethylcarbazole (aec)/h 2 o 2 as substrate. an overview of all constructed vsv vrp is provided in table 1 . marc-145 cells grown on 12-mm-diameter cover slips were inoculated for 90 min at 37 °c with recombinant vrp using a multiplicity of infection (moi) of 3 iu/cell. at 6 h post infection, the cells were fixed with 3% pfa for 20 min and washed with pbs containing 0.1 m (w/v) glycine. the cells were permeabilized with 0.25% (v/v) triton x-100 for 5-10 min and subsequently incubated with primary and secondary antibodies, diluted in pbs containing 1% bovine serum albumin (bsa). after each incubation period (60 min, room temperature), the cells were washed three times with pbs. finally, the cells were washed with distilled water and embedded in mowiol 4-88 mounting medium (sigma-aldrich). all pigs were obtained from the specific pathogen free (spf) breeding facility of the institute of virology and immunology ivi. three experiments were performed. for each experiment the pigs were randomly assigned to treatment groups housed in separate stables. the groups were immunized by intramuscular injection of 5 ml of cell culture supernatant containing 10 7 -10 8 iu/ml recombinant vrp. in the first experiment, 2 groups of pigs (n = 5) were immunized three times at 9½, 13½ and 18½ weeks of age with vsvδg(gp5/m) or with the control vsv*δg vrp, respectively. in the second experiment, one group of pigs (n = 4) was immunized twice at 10½ and 14½ weeks of age with a mixture of vsvδg(gp5/m) and vsvδg(gp4/gp3/gp2) and the second group (n = 3) received the control vsv*δg vrp. in the third experiment, 4 groups of pigs (n = 2) were immunized three times at 5, 6 and 7 months of age with two different vsv vrp each injected at two different sites, i.e. vsv*δg(n) and vsv*δg(ha belz ), vsv*δg(gp3ecto-myc) and vsv*δg(m2 belz ), vsv*δg(gp4-ha) and vsv*δg(na belz ), vsv*δg(control) and vsv*δg(np belz ), respectively. in all experiments, the pigs were challenged via the intranasal route with 10 6 tcid 50 /animal of the prrsv strain olot/91 3-4 weeks after the last vaccination. the challenge virus was diluted in 5 ml mem and administered dropwise intranasally. blood was taken before each vaccination and at regular intervals after vaccination and after the challenge infection. serum was stored at −70 °c. body temperature and clinical score were monitored daily according to a defined scoring system [55] . the experiments in pigs were performed in compliance with the swiss animal protection law and approved by the animal welfare committee of the canton of berne, switzerland (authorization number be89/11). titration by end point dilution was performed in marc-145 cells grown in 96-well plates. the cells were inoculated with tenfold serially diluted serum samples. at 48 h pi, the cells were fixed and immunoperoxidase staining with the anti-n mab 13e2 was performed according to standard protocols. the 50% end point titre was expressed as tissue culture infectious dose 50% (tcid 50 )/ ml, with a limit of detection of 1.7 log 10 tcid 50 /ml. total cellular rna was extracted from pig sera using the nucleospin multi 96 virus kit (macherey-nagel) on a freedom evo robot (tecan) and stored at −70 °c. for detection of viral rna, a reverse transcriptase quantitative pcr (rt-qpcr) based on the amplification of the conserved region of orf7 was performed in duplicates employing gfp messenger rna as internal control [56] . the results were expressed as the total number of cycles minus the quantification cycle (cq)-value. the elisa prrs x3 (idexx laboratories) was used for measuring anti-prrsv igg antibodies. samples to positive (s/p) ratios higher than 0.4 were considered positive. antibodies against prrsv gp5, gp4 and gp3 were detected using in house competitive (gp5) and indirect (gp4 and gp3) elisas (ingenasa, madrid, spain). these elisas were performed with plates coated with recombinant gp5, gp4 and gp3 antigens from prrsv olot/91. for the gp5 elisa, the percentage of competition was calculated using the formula [(odneg − odsample)/(odneg − odpos)] × 100. positive and negative controls were provided by ingenasa (spain). samples were considered positive if the percentage of competition was >37%. for the indirect elisas, appropriate cut-offs were set. for the detection of c-reactive protein (crp; genway), haptoglobin (hp; genway) and ifn-γ (mabtech) in pig sera, commercially available elisas were used according to the manufacturer's protocols. porcine interferon-α (ifn-α) was determined by elisa as described previously [57] . serum samples were heat inactivated at 56 °c for 30 min prior to performing the serum neutralization assay. 50 µl of two-fold serially diluted sera were mixed with an equal volume of 100 tcid 50 of prrsv olot/91 and incubated in 96-well tissue culture plates for 1 h at 37 °c. after 1 h, 10 4 marc-145 cells were added to each well. after 6 days, the culture plate was fixed with 3% pfa. the presence of virus was detected by cpe and by staining with the anti-n mab and aec/h 2 o 2 . the virus neutralization titre was expressed as the reciprocal of the serum dilution leading to 50% reduction of infection. data were analysed with the graphpad prism 6.01 software. significant differences between groups were assessed by multiple t tests. p < 0.05 was considered significant. in previous work, a propagation-incompetent vsv replicon vector was generated by replacing the gene of the glycoprotein g in the fourth transcription unit of the viral genome with influenza virus genes. this replicon contained the gfp gene [referred to by an asterisk (*) in the vector nomenclature] in an additional transcription unit at position 5 [49] . based on this vector, we generated several vsv vrp expressing the individual structural antigens of prrsv olot/91 or combinations thereof for evaluation as prrs vaccine candidates (see "materials and methods"; table 1 ). the empty parental vsv*δg vector served as control [49] . the vsv vrp-mediated expression of the recombinant prrsv antigens was studied in marc-145 cells, taking advantage of the broad tropism of the vsv particles. this allowed direct comparison of the recombimarc-145 cells infected with either vsv*δg(gp3-flag) or vsvδg(gp4/gp3/gp2) reacted specifically with the anti-gp3 mab, while cells infected with either vsv*δg(gp4-ha) or vsvδg(gp4/gp3/gp2) reacted with a polyclonal anti-gp4 immune serum (figure 2 ). since a specific antibody against gp2 was not available, the expression of gp2 could not be demonstrated. any tag was omitted on purpose in the vsvδg(gp4/gp3/ gp2) to preserve the natural conformation of the three proteins. nevertheless, myc-tagged gp2 expression was detected from vsv*δ(gp2-myc) using the anti-myc serum (not shown). with the aim of evaluating whether protein secretion into the supernatant may enhance b-cell responses, the vsv*δg(gp3ecto-myc) replicon was constructed for the expression of the ectodomain of gp3 fused to a igκ leader sequence and an optimal signalase cleavage site. the myc-tagged ectodomain of gp3 reacted with the anti-gp3 mab and anti-myc serum as expected ( figure 3a ). western-blot analysis with the anti-myc serum demonstrated the presence of a 35 kda protein in the cell lysate (ly) which was however missing in the supernatant (sn) of vsv*δg(gp3ecto-myc) infected marc-145 cells ( figure 3b ). this showed that the ectodomain of gp3 was retained in intracellular compartments despite the igκ signal sequence and the lack of the transmembrane domain. finally, expression of the prrsv n antigen by vsv*δg(n) was demonstrated by immunofluorescence ( figure 4a ) and by western blot ( figure 4b ) using the anti-n mab. a 16 kda protein was detected in the lysate of both, vsv*δg(n)-and prrsv olot/91-infected cells. the gp5 and m complex constitutes the major protein component of the prrsv envelope against which neutralizing antibodies are formed [18, 19, 26] . therefore, we first determined the immunogenicity of gp5/mrecombinant vsv replicons in pigs. two groups of five pigs were immunized three times with vsvδg(gp5/m) or with the control vsv*δg respectively, and were challenged 3 weeks later with the homologous prrsv olot/91 strain. following fever (40.3-41.5 °c) on day 2 pi, whereas the control pigs had no fever despite slightly elevated body temperature ( figures 5a and b) . all animals developed a low viremia of short duration ( figures 5c and d) . a maximum mean virus titre of 3.0 log 10 tcid 50 /ml was reached in the serum at day 2 post challenge. the virus was detectable up to day 8 and viral rna up to day 13 after challenge. no significant differences in viremia and viral rna in serum were observed between the vaccinated group and the control group at any time. before challenge infection, vaccination with vsvδg(gp5/m) did not induce any detectable antibody response against gp5 as measured by elisa (not shown). the first gp5-specific antibody responses were detected by elisa in the vsvδg(gp5/m)-vaccinated group in 2 out of five pigs on day 6 after the challenge, and all pigs of this group were positive on day 13 pi. all pigs of the vsv*δg control group remained gp5 antibody negative at this time (table 2 ). in the vsv*δg control group, gp5specific antibodies were detected for the first time on day 21 pi in 2 out of the 5 pigs. seroconversion against n in response to the prrsv challenge infection was similar in the two groups, with the first n-specific seroconversion observed on day 10 pi (table 2) . virus neutralizing antibodies were not detected in any of the animals, neither before nor after the challenge (day 0 and day 21 pi). ifn-α and ifn-γ could not be detected in the serum at any time (data not shown). specific t-cell responses were not detected before challenge (not shown) and were therefore not further investigated. the acute phase proteins crp and hp which are early and sensitive markers of disease and inflammation including prrsv infection [58] were increased in both groups between days 2 and 6 pi ( figures 5e and f) . however, no significant differences could be detected between vaccinated and control animals at any time. thus, apart from priming pigs for gp5specific antibody responses after challenge infection, the vsvδg(gp5/m) vector could not induce any seroconversion against gp5 before challenge nor any sign of protection from viremia after infection. in order to determine whether the immune responses could be enhanced and induced before challenge by providing the three minor glycoproteins gp4, gp3 and gp2 together with gp5 and m, pigs (n = 4) were immunized twice with a mixture of vsvδg(gp5/m) and vsvδg(gp4/gp3/gp2) or with vsv*δg as control (n = 3) followed by prrsv olot/91 challenge. on day 2 after challenge, two out of the four vaccinated pigs developed mild fever (40 °c; figure 6a ) while all control animals remained free of fever (<40 °c; figure 6b ). all animals developed a short viremia following challenge infection with no significant differences in viral rna load and virus titres between the vaccinated and control groups (figures 6c and d) . as in the previous experiment, a maximum mean virus titre of 3.0 log 10 tcid 50 /ml was reached at day 2 post challenge. again, before challenge infection, vaccination with vrp expressing the structural proteins of prrsv did not induce any detectable antibody response against gp5, gp4 and gp3 as measured by elisa (not shown). following challenge infection, gp5-specific antibody responses were detected on day 7 pi in 2 out of 4 pigs, and all pigs vaccinated with vrp expressing the prrsv proteins seroconverted to gp5 at day 11 pi (table 3 ). all pigs of the vsv*δg control group remained negative until day 24 pi. seroconversion to gp3 (table 3) vsv*δg control group remained negative until day 24 pi. a gp4-specific immune response could be detected only in 2 out of 4 vaccinated animals on day 24 pi. here also, the seroconversion against n following challenge was similar in the two groups ( table 3) . none of the animals developed any neutralizing antibodies before nor after challenge (day 0 and day 24 pi). ifn-α and ifn-γ were not found in any of the sera in this experiment either (data not shown) whereas the serum crp and hp levels increased significantly in both groups between days 2 and 4 after challenge and declined subsequently. on day 4 pi, the crp and hp levels were significantly different between the vaccinated and the control group ( figures 6e and f) , suggesting that the vaccinated group developed a slightly reduced inflammatory response after co-vaccination with vsvδg(gp5/m) and vsvδg(gp4/ gp3/gp2). together, these two vaccination trials show that vsv vrp cannot induce any detectable seroconversion against prrsv before challenge nor any protection against virus infection and viremia. nevertheless, these vectors can clearly prime pigs for earlier prrsv-specific antibody responses after challenge infection. the poor immunogenicity of vsv vrp expressing prrsv envelope proteins contrasts with previous reports showing protection against iav and btv infections in chickens and sheep, respectively [49, 50, 52] . thus, the lack of antibody induction after immunization of pigs with vrp expressing the five prrsv envelope proteins raises the questions whether this vector vaccine is suitable for induction of antibody responses in pigs or whether the prrsv proteins are poorly immunogenic per se. in order to test this, the immunogenicity of prrsv and iav proteins expressed from vsv vrp was assessed in the same animal by co-vaccination with two different vsv vrp constructs expressing a prrsv protein (n, gp3ecto, gp4 or empty vector) and a iav protein (ha, m2, na, np), respectively, injected at two different sites (see "materials and methods"). after 3 vaccinations, the pigs were challenged with the homologous prrsv olot/91 strain. while, antibody responses were detected against all influenza virus proteins on day 14 after vaccination (including neutralizing antibodies, not shown), no seroconversion against any of the prrsv proteins except n could be detected before challenge (table 4 ). pigs vaccinated with vsv*∆g(n) seroconverted on day 63 after vaccination (7 days after the third vaccination). following challenge infection with prrsv olot/91, the control group seroconverted on day 21 pi. there was no evidence of protection from virus infection after prrsv challenge as indicated by the kinetics of viremia (data not shown). nevertheless, a gp3-specific immune response was induced earlier after challenge (day 14 pi) in the vsv*δg(gp3ecto-myc) vaccinated pigs compared with the vsv*δg-vaccinated pigs (no response on day 21 pi). a gp4-specific immune response was also induced on day 14 pi whereas the vsv*δg-vaccinated pigs stayed seronegative against gp4. in line with the two previous immunization trials, these data show again a priming effect. more importantly however, simultaneous vaccination of the same pigs with two different vrp show clearly that the prrsv antigens are far less immunogenic than any of the iav antigens tested. the current prrsv vaccines on the market are of limited efficacy and come with several drawbacks [32] . avoid the biological risks of virus spread and to circumvent potential immune modulating properties of modified live prrsv vaccines [40] . therefore, we explored the immunogenic and protective potential of vsv replicon particles as a vectored vaccine approach against prrsv. such vrp were successfully used before as efficacious experimental vaccines against sars, iav and btv [48] [49] [50] [51] [52] . in the present study, vsv replicons were engineered to express prrsv structural proteins, individually or in combination (table 1) . emphasis was on gp5, m, gp4 and gp3, since these proteins are important targets for neutralizing and protective antibodies [11, 23, 26, 27] , with the major neutralizing epitopes residing on gp5 [18] . since several studies have shown that co-expression of prrsv antigens resulted in better humoral and cellular immune responses than expression of the individual proteins [30, 60] , gp5, m, gp4, gp3 and gp2 proteins were co-expressed to partly mimic the formation of the gp5/m and gp4/3/2 oligomers. vaccination of pigs with vrp expressing two or five prrsv envelope proteins in total did not result in any significant reduction of viremia compared with the control group. a tendency for reduced prrsv-related inflammatory responses was observed only when all five proteins were expressed. interestingly, vaccination with gp5/m induced fever up to 41.5 °c for 1 day following challenge infection with prrsv olot/91 whereas mockvaccinated pigs remained asymptomatic. fever was less pronounced when all envelope proteins were included in the vaccine. enhanced disease following vaccination with recombinant gp5 and m was observed previously [42, 61] . this was attributed to antibody-dependent enhancement of disease during natural infection, which is probably mediated by non-neutralizing antibodies [26, 62] . such antibodies were shown to increase virus infection in porcine alveolar macrophage cultures and in vivo [62] involving different fcγr isoforms [63, 64] . neutralizing antibodies were not found at any time following vaccination and challenge infection. nevertheless, the vaccinated pigs seroconverted 15-17 days earlier than the mock-vaccinated animals after challenge virus infection, indicating that immunization with the recombinant vrp primed the immune system. in naïve pigs, the delayed seroconversion against the envelope proteins as opposed to the n protein after prrsv infection or after immunization with vectored vaccines is well table 3 documented [31, 40, 42, 59] . of note, with the marc-145-adapted olot/91 virus used in this study, seroconversion against n became detectable 9-14 days after challenge only (tables 2, 3 , 4) as opposed to the reported occurrence of anti-n antibodies as early as 5 to 7 days after prrsv infection [31, 65] . this is probably related to the low level and short duration of replication of the marc-145-adapted virus in pigs ( figures 5c, d, 6c, d) . despite the poor replication of this virus in vivo, vaccination of pigs with vrp did not result in any significant reduction of viremia. a better immunogenicity of prrsv proteins was reported when the antigens were modified, coupled to immunostimulatory molecules or administered as purified proteins along with adjuvants [66, 67] . this raises the question whether the immunogenicity of the prrsv envelope proteins expressed from vrp is affected by glycosylation, subcellular localization, intracellular retention or low stability. in order to elaborate on this, gp3 was modified with the aim of obtaining secreted gp3 by replacing the leader sequence with a canonical igκ signal sequence followed by an optimal signalase cleavage site and by deleting the hydrophobic transmembrane anchor. however, the modified gp3 was retained in the cell, and no seroconversion against gp3ecto was obtained in absence of challenge virus infection. the lack of gp3 secretion may be related to the fact that gp3 is naturally retained in intracellular compartments despite the n-terminal signal sequence [11] . in order to address the question whether prrsv structural proteins are immunogenic at all when expressed by vsv vrp in pigs, a vector expressing prrsv n protein was included in one vaccination trial. n was chosen because it is the most immunogenic structural protein after prrsv infection [31, 65] . indeed, vsv*δg(n) was the only vrp capable of inducing a detectable antibody response in the absence of a viral challenge ( table 4 ). the co-vaccination experiments with vrp expressing iav and prrsv envelope proteins corroborated these results. the iav proteins were strongly immunogenic while the prrsv envelope proteins were not, clearly demonstrating that the vsv vector per se is functional in pigs. although several vector vaccines failed to induce a protective immune response, immunizations essentially primed the immune system to a challenge infection [41, 42] . these reports and the present study altogether suggest that the prrsv envelope proteins are expressed in a way to hide partially from the immune system. whether this immune evasion strategy is due solely to glycan shielding [68] or to a particular intracellular topology remains to be investigated. future efforts in vectored vaccine development should consider these aspects to enhance the immunogenicity of the prrsv antigens. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection porcine reproductive and respiratory syndrome porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states origin of highly pathogenic porcine reproductive and respiratory syndrome virus pathogenesis and antigenic characterization of a new east european subtype 3 porcine reproductive and respiratory syndrome virus isolate nidovirales: evolving the largest rna virus genome the ever-expanding diversity of porcine reproductive and respiratory syndrome virus molecular evolution of prrsv in europe: current state of play arterivirus molecular biology and pathogenesis membrane proteins of arterivirus particles: structure, topology, processing and function discovery of a small arterivirus gene that overlaps the gp5 coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses intracellular synthesis, processing, and transport of proteins encoded by orfs 5 to 7 of porcine reproductive and respiratory syndrome virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus the minor envelope glycoproteins gp2a and gp4 of porcine reproductive and respiratory syndrome virus interact with the receptor cd163 immunological responses of swine to porcine reproductive and respiratory syndrome virus infection the amino acid residues at 102 and 104 in gp5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody role of neutralizing antibodies in prrsv protective immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain the primary gp5 neutralization epitope of north american isolates of porcine reproductive and respiratory syndrome virus gp4-specific neutralizing antibodies might be a driving force in prrsv evolution posttranslational processing and identification of a neutralization domain of the gp4 protein encoded by orf4 of lelystad virus a variable region in gp4 of european-type porcine reproductive and respiratory syndrome virus induces neutralizing antibodies against homologous but not heterologous virus strains heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection categorization of north american porcine reproductive and respiratory syndrome viruses: epitopic profiles of the n, m, gp5 and gp3 proteins and susceptibility to neutralization characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus co-expressing gp5 and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (prrsv) pathogenesis of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) taming prrsv: revisiting the control strategies and vaccine design porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction impact of genetic diversity of european-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv vectored vaccines to protect against prrsv antigenic structures stably expressed by recombinant tgev-derived vectors immunization with dna vaccines containing porcine reproductive and respiratory syndrome virus open reading frames 5, 6, and 7 may be related to the exacerbation of clinical disease after an experimental challenge vaccine production in plant systems: an aid to the control of viral diseases in domestic animals: a review baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain olot/91. involvement of orf3 and orf5 proteins in protection immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing gp5/gp3 of porcine reproductive and respiratory syndrome virus and swine il-18 ctla4 mediated targeting enhances immunogenicity against prrsv in a dna prime/killed virus boost strategy use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector a recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (h7n1) vaccination with recombinant rna replicon particles protects chickens from h5n1 highly pathogenic avian influenza virus vesicular stomatitis virus vectors expressing avian influenza h5 ha induce cross-neutralizing antibodies and long-term protection vesicular stomatitis virus replicon expressing the vp2 outer capsid protein of bluetongue we thank luis enjuanes, centro nacional de biotecnología, madrid, spain, for supplying the marc145-adapted prrsv olot/91 strain, the cdna for orf2 to 6 and the rabbit anti-gp4 serum; we thank also hans nauwynck, university ghent, belgium for providing the mabs 13e2 and vii2d, and ingenasa, madrid, spain for the mabs 11e10c7 and 3ah9. we thank markus gerber and nuria de la roja for excellent technical assistance, and daniel brechbühl and veronika ayala for animal care. the authors declare that they have no competing interests. authors' contributions nr, kcm, gz and as conceived the study. me, gz, nr, as, mgd and mjr designed the experiments. me performed most of the experiments. mgd, js and mjr performed the glycoprotein-specific elisas. sl and mer contributed with iav vrp. me, nr, gz and mgd wrote the manuscript. all authors read and approved the final manuscript. key: cord-327199-ggomuomb authors: moerdyk-schauwecker, megan; hwang, sun-il; grdzelishvili, valery z. title: cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions date: 2014-08-08 journal: plos one doi: 10.1371/journal.pone.0104688 sha: doc_id: 327199 cord_uid: ggomuomb virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. while many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. vesicular stomatitis virus (vsv) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. using mass spectrometry, we previously examined cell type dependent host protein content of vsv virions using intact (“whole”) virions purified from three cell lines originating from different species. here we aimed to determine the localization of host proteins within the vsv virions by analyzing: i) whole vsv virions; and ii) whole vsv virions treated with proteinase k to remove all proteins outside the viral envelope. a total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in proteinase k treated virions. most of these proteins have not been previously shown to be associated with vsv. functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. using western blotting, the presence of several host proteins, including some not previously shown in association with vsv (such as yes1, prl1 and ddx3y), was confirmed and their relative quantities in various virion fractions determined. our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of vsv. vesicular stomatitis virus (vsv, family rhabdoviridae) is a prototypic nonsegmented negative-strand rna virus (order mononegavirales) serving as a model for other human and animal pathogens including other rhabdoviruses such as rabies virus. vsv is also being widely investigated as a vector for vaccines (reviewed in [1] ), gene therapy (reviewed in [2] ) and oncolytic (anticancer) virotherapy (reviewed in [3, 4] ). as a result, currently two phase i human clinical trials evaluating the safety of the vsv-based hiv vaccines (clinicaltrials.gov, trials nct01438606 and nct01578889) are currently in progress. in addition, a phase i human clinical trial using replication-competent oncolytic vsv against hepatocellular carcinoma is in progress (trial nct01628640). vsv replicates in the cytoplasm and contains a nonsegmented, negative-strand rna genome of approximately 11 kb encoding five viral proteins, all of which are included in the mature virion. the large polymerase protein (l) and phosphoprotein (p) together form the viral rna dependent rna polymerase (rdrp). in mature virions, the rdrp is associated with the nucleocaspid (n) protein encapsidated viral genome, together forming the ribonucleoprotein (rnp) complex. the matrix (m) protein is responsible for nucleocapsid condensation and is the primary driving force behind viral budding through the host cell plasma membrane, whereby the virion obtains its lipid bilayer envelope. embedded in that envelope is the transmembrane glycoprotein (g), which is essential for receptor binding and cell entry (reviewed in [5] ). vsv virions, like those of many viruses, contain not only virus encoded-proteins, but also host (cellular) proteins [45] . some of these proteins are enclosed within the virion structure, while others, in the case of enveloped viruses like vsv, are embedded in the host-derived membrane [6] . while many of these incorporations could be ''accidental'', others may reflect the mechanism of virus assembly, be necessary for viral function, impact host range and infection efficiency, or influence the outcome of subsequent infections. for example, a recent study showed that depletion of a number of host proteins normally incorporated into herpes simplex virus type-1 virions not only reduced virion production in those cells, but that new infections with the resulting virions also yielded fewer progeny virions even in cells expressing normal levels of the host protein [7] . the host-derived proteins of a virus may also affect the host immune response. this is an especially important consideration for viruses, including vsv, used in therapeutic applications where large numbers of virus particles are administered, as it may influence efficacy as well as the potential for adverse side effects. incorporation of icam-i into the envelope of human immunodeficiency virus type-1 (hiv-1) not only increased infection efficiency [8, 9, 10, 11] but also interfered with virus neutralization by host antibodies [12, 13, 14, 15] . in another example, the presence of host complement control proteins such as cd46, cd55 and cd59 in the viral envelope has been shown to protect against antibody dependent complement mediated virus lysis in several viruses including human t cell leukemia/ lymphoma virus type i [16] , human cytomegalovirus [16] , hepatitis c virus [17] , hiv-1 [18, 19] , extracellular enveloped vaccinia virus [20] , simian virus 5 [21] and mumps virus [21] . to comprehensively look at host protein incorporation, a number of purified viruses have been analyzed by mass spectrometry including poxviruses [22, 23, 24] , herpesviruses [25, 26, 27, 28, 29, 30, 31, 32] , orthomyxoviruses [33] , coronaviruses [34, 35] , retroviruses [36, 37, 38, 39] , paramyxoviruses [40, 41] , baculoviruses [42] , hytroviruses [43] and arteriviruses [44] . our previous study examined vsv virions grown in three different cell lines (bhk-21, 4t-1 and a549) originating from different species, and found a number of similarities and differences in the host protein content [45] . here we conduct an analysis not only of intact (''whole'') virions, but also virions treated with proteinase k (prok) to remove surface proteins to look at the localization of host protein incorporation into the virion. syrian golden hamster kidney fibroblast cells (bhk-21; atcc# ccl-10) were grown in monolayer cultures maintained in minimum essential medium (eagle's mem, cellgro) supplemented with 9% fetal bovine serum (fbs, gibco), 0.3% glucose (w/v), 3.4 mm l-glutamine, 90 u/ml penicillin and 90 mg/ml streptomycin, and kept in a 5% co 2 atmosphere at 37uc. infectivity [plaque forming units (pfu) per ml] of virus stocks was determined by standard plaque assay on bhk-21 cells. recombinant wild-type (wt) vsv (indiana serotype) was generated previously [46] using pbs-l, pbs-p, pbs-n, and pvsvfl(+) plasmids, and was kindly provided by john k. rose (yale university) [47] . to grow and purify virus vsv, bhk-21 cells were infected with at a multiplicity of infection (moi) of 0.001 and incubated at 37uc in media containing 5% fbs. virus containing media was collected around 24 hours (h) post infection (p.i.) when most cells were infected but significant cell detachment had not yet occurred in order to maximize exclusion of cellular debris. the media was centrifuged at 3,0006 g for 10 minutes (min) to remove large cellular debris. the virus was then purified as described in [48] , with slight modifications. in brief, clarified supernatants were underlayed with 5 ml 20% (w/v) sucrose in hen buffer (10 mm hepes ph 7.4, 1 mm edta, 100 mm nacl) and centrifuged at 28,000 rpm and 4uc for 3.5 h in a beckman sw32 ti rotor. the resulting virus-containing pellet was resuspended overnight in hepes buffered saline, ph 7.5 [hbs; 21 mm hepes, 140 mm nacl, 45 mm kcl, 0.75 mm na 2 hpo 4 , 0.1% (w/v) dextrose] and then centrifuged in a 7.5-27.5% continuous gradient of optiprep (axis shield) in hbs at 26,500 rpm and 4uc for 30 min using a beckman sw40 ti rotor. the virus-containing band was removed from the gradient, diluted with et buffer (1 mm tris-hcl ph 7.5, 1 mm edta), pelleted by centrifugation at 27,000 rpm and 4uc for 1.5 h using a beckman sw40 ti rotor and resuspended in et buffer. for protease treatment, purified virions were treated with 0.08 mg prok per 1 mg total protein, re-purified by centrifugation through a sucrose cushion as previously described [45] and resuspended in et buffer. for isolation of viral rnp complexes, purified virions not treated with prok were disrupted as previously described [49] . in brief, virions were disrupted in a buffer containing a final concentration of 10 mm tris-hcl (ph 8.0), 5% glycerol (v/v), 0.4 m nacl, 1.85% triton x-100, and 0.6 mm dtt, and pelleted through a 30% glycerol cushion onto a 100% glycerol cushion by centrifugation at 38,000 rpm and 4uc for 2 h using a tla 100.2 rotor. under these conditions, proteins with moderate to high affinity for the viral nucleocapsid, including the viral l/p polymerase complex, remain associated with the nucleocapsid [49] . the recovered rnp complexes were diluted 1:1 (v/v) with a buffer containing 50 mm tris-hcl (ph 8.0), 10 mm nacl, 5 mm mgcl 2 and 2 mm dtt. virions were absorbed to carbon-formvar coated grids (electron microscopy sciences) by floating grids on 5 ml drops of sample for 30 seconds (s). grids were blotted dry, stained with 2% uranyl acetate in water for 30 s, blotted to remove excess stain and airdried. samples were visualized using a jeol jem 2100 lab6 transmission electron microscope. total protein equaling 50 mg for purified virion samples and 60 mg for prok treated virions was separated on a 10% tris-glycine sds-page gel under reducing conditions and stained with coomassie brilliant blue r250. these quantities were chosen so that the total amount of viral n plus p protein was approximately equivalent for both sample types. each gel lane was cut into 13 or 14 gel bands for analysis. gel pieces were subjected to in-gel trypsin digestion and the resulting peptides were extracted from the gel matrix, separated using waters acquity ultra performance liquid chromatography (uplc) with nano-split and analyzed using a ltq-xl tandem mass spectrometer (thermofisher scientific, waltham, ma) as described previously [50] . briefly, samples were separated by a 65 min linear gradient from 95% solvent i (0.1% formic acid in water)/solvent ii (0.1% formic acid in acetonitrile) to 50% solvent i/ii at a flow rate of 500 nl/min on reversed phase chromatography using a trap/elute method with a in-house c 18 sample trap in line with a c 18 analytical column. each full ms scan was followed by eight ms/ ms scans of the most intense ions with data-dependent mode using the dynamic exclusion option (top 8 method). the spectra were searched using the sequest algorithm of the bioworks software (thermofisher, san jose, ca; version 3.3.1 sp1) against the ipi.human.v.3.18 and ipi.mouse.v.3.18 databases concatenated with a vsv and sendai virus protein database. a parent ion mass tolerance of 2.0 da, fragment ion mass tolerance of 1.0 da, and a 16 da differential modification for methionine oxidation were used for search parameters. protein identifications were accepted when the peptide probability was greater than 95.0% [51] , the protein probability was greater than 99.0%, and contained at least two unique peptides. scaffold software was used for data compiling of each group and calculating spectral counts, unique peptides, percent coverage and empai [52, 53, 54] . while a single set of gel bands was isolated for each sample, three replicate uplc-ms/ms runs were conducted for the whole and prok virion samples, although one set of runs could not be used for the whole virions due to poor data quality. protein enrichment was analyzed using the database for annotation, visualization and integrated discovery (david) v.6.7 [55, 56] . using the mus musculus genome as the background data set, protein sets were analyzed for enrichment using the terms from the gene ontology (go) biology process, cellular component or molecular functions fat databases. the go fat databases contain the more specific go terms while excluding the more general terms. the enriched terms were then subjected to cluster analysis using the default settings, to identify groups of related enriched terms, with overall enrichment scores based on the ease scores of the member terms. the broadest term, representing most if not all the proteins in the cluster, is used here to describe the cluster. cellular lysates were prepared by mock infecting bhk-21 cells or by infecting them with vsv at a moi of 0.05. cells were harvested at 18 h p.i. and lysed in ripa buffer (25 mm tris-hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate and 0.1% sds). protein concentrations of cellular lysates, purified virions, prok treated virions and rnp complexes were determined by bradford assay. 50 mg of purified virions, 60 mg prok treated virions, 15 mg of rnp complexes and 10 mg of cellular lysates were separated on tris-glycine 10% sds-page gels, transferred to pvdf membranes and rapidly stained with the reversible dye ponceau s prior to the use of antibodies to confirm viral protein loading and the quality of protein transfer from gel to membrane. membranes were blocked in tbs (0.5 m nacl, 20 mm tris ph 7.5) with 0.1% tween 20 and 5% non-fat milk powder and then probed with antibodies against cc2d1a (a300-285a; bethyl laboratories), yes1 (3201; cell signaling), itch (32/itch; bd transduction laboratories), hsc70 (k-19; santa cruz biotechnology), prl2 (05-1583; millipore), ck1 (2655; cell signaling), or ddx3y (pa5-22050; thermo scientific). detection was with species-specific horseradish peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence plus (ecl+) protein detection system (ge healthcare) and captured using a chemidoc-it imaging system (uvp imaging, upland ca). recombinant wt vsv was grown in bhk-21 cells and purified using gradient centrifugation. the purity of the resulting material was then examined by electron microscopy (em). as seen in figure 1 , nearly all of the material present was clearly identifiable as vsv virions, although many of them were bent, a previously observed form that is generally believed to be infectious [57] and may represent an em processing artifact [58, 59] . the titer of these purified virions on bhk-21 cells was 1.4610 11 pfu/ml, demonstrating this preparation was highly infectious. a portion of these whole virions was used directly for proteomic analysis and additional confirmation assays, while the other portion was treated with prok and re-purified (fig. 2) . prok treatment cleaves all proteins on the exterior of the virions as well as the extracellular domain of all transmembrane proteins. however, it cannot penetrate the viral envelope, leaving all vsv proteins except for g intact, as well as all host proteins contained within the virion and the transmembrane and cytoplasmic domains of the host membrane proteins located in the viral envelope. this treatment also tends to alter the density of any remaining cell derived vesicles relative to the treated virions, facilitating their removal during the subsequent repurification step [60] . however, proteins on the interior of any residual cellular vesicles would still be detectable. for mass spectrometry (ms) analysis, total protein from 50 mg purified virions or 60 mg prok treated virions was separated by 1d-sds-page. these quantities were chosen so that the amount of viral n and p protein was approximately equal in both sample types as determined by coomassie staining (fig. 3a) to facilitate comparisons between samples. importantly, while n, p and l bands were similar in both virion preparations, no full length g protein was visible for prok treated virions, indicating the prok treatment was highly successful. after separation by sds-page, the resolved proteins were cut out in a series of bands as indicated in figure 3a . these bands were then subjected to in-gel trypsin digest and the resulting peptides were extracted, separated by uplc and analyzed by tandem ms (ms/ms). in this study, virions were grown on bhk-21 cells as it is the standard cell line for growth of vsv and the high virion yields aid in purification. however, a complete database of syrian hamster proteins is not available. instead, peptides were identified by matching them to either a human or a mouse database. the results from both searches were highly similar; therefore only those from the search of the mouse database are presented here. in addition to all five viral proteins, 257 different host proteins were identified in total (table s1) , with 181 proteins identified in whole virions and 183 proteins identified in prok treated virions (fig. 3b ). this is a considerably larger number of host protein than identified in our previous study where 30 proteins were identified in a different preparation of bhk-21 derived virions [45] . this is likely primarily due to dividing the samples into a larger number of bands following 1-d sds-page. in addition, in the present study we used enhanced sample separation through use of uplc, which should also allow for more sensitive detection. of the 30 proteins detected in bhk-21 derived whole virions in our previous study, 27 were detected in at least one sample type here, showing consistency in the proteins detected in independent virion preparations purified using two different methodologies (continuous iodixanol gradient versus discontinuous sucrose gradient). differences in the purity of the virions prepared using these two methods could also contribute to differences in the number of host proteins identified. however, em images, staining of total protein on sds-pages gels and infectivity (4.2610 7 pfu/mg total protein for the present method versus 2.4610 7 pfu/mg total protein for our previously published method [45] ) all indicate virions prepared using the method presented here are of equal or greater purity to those used previously. surprisingly, more than 40% of proteins found in prok treated virions were not found in whole virions even though the prok treated virions were derived from the whole virion preparation (fig. 2) . one possibility is that some of these proteins may have been masked by more abundant host or viral proteins that were removed by treatment. for example, 25% (19/76) of the proteins detected in prok treated virions but not whole virions were from the position correlating to that of the viral g protein in the whole virion sample (table s1 and fig. 3a) . another possible explanation is that these proteins are in low abundance or possess other properties making them difficult to detect consistently by ms. of the proteins found only in prok treated virions, 50% of proteins were identified based on detection of only two unique spectra (the minimum number allowed under our criteria), as opposed to 15% for proteins also identified in whole virions (table s1 ). unsurprisingly, ease of detection also seemed to be a major determinate of consistency of detection between technical replicates. proteins found in all technical replicates were identified based on only two unique spectra in 19% and 17% of cases for whole virions and prok treated virions respectively, while proteins found in only one technical replicate were identified based on only two spectra in 71% and 77% of cases (fig. 3c-d and table s1 ). in general, the position of the identified proteins on the gel was consistent with the predicted molecular mass of the proteins (table s1 and fig. 3a) although some appeared at higher molecular masses, likely due to post-translational modifications affecting protein mobility. exceptions to this general pattern were keratins, common environmental contaminants, which were found across a wide range of molecular weight and therefore excluded from this analysis. following prok treatment, 74 of the 181 proteins identified in whole virions were no longer detectable, likely due to their removal by the treatment. however, other proteins were still detected in the prok treated sample but appeared at a lower molecular mass versus the predicted molecular mass and/or that observed for whole virions, likely due to the removal of the extracellular domain. for example, hepatocyte growth factor receptor was found in a slice spanning approximately 140-225 kda in the whole virion while it was found in slices spanning approximately 50-80 kda in prok treated virions. this demonstrates the value of information about the approximate size of the proteins in the sample (as determined by the position of the gel band) in interpreting data from proteinase treated samples. although the primary focus of this study was determining host protein incorporation and localization in vsv virions, our search database also included vsv protein sequences allowing for their detection. in untreated samples, a mean of 1231-182 spectra per technical replicate were detected for each of the five vsv encoded proteins (fig. 4a) . the number of spectra went up upon prok treatment for all vsv proteins except g where there was a sharp reduction. this is consistent with our other data (fig. 3a) demonstrating that prok treatment is effectively removing the extracellular domain of the vsv g protein. removal of the extracellular domain was confirmed by determining the distribution of the spectra and peptides derived from the g protein (fig. 4b) . the mean number of spectra associated with the transmembrane domain and cytoplasmic tail of the g protein were unchanged between whole and prok treated virions, while prok treatment decreased the number of spectra associated with the extracellular domain of g. in looking at the distribution of the detected peptides in the g protein, prok treatment completely eliminated peptides associated with the n-terminal portion of the extracellular domain while a few were still detected in the more c-terminal portion of the domain, suggesting this region has a slight degree of resistance to proteinase cleavage, although the decrease in spectral counts suggests the majority the g proteins were cleaved. while ms analysis of unlabeled proteins cannot be used to make quantitative comparisons of protein abundance, spectral counts or measures derived from spectral counts (including empai values shown in table s1 ), can be used to make semi-quantitative comparisons [61] . based on spectral counts, few if any of the host proteins come close to matching the viral proteins in abundance within the virion. only one host protein, low-density lipoprotein receptor-related protein 1 (lrp1), had a mean of more than 100 total spectra per technical replicate in untreated whole virions, while only 17 (9.4%) had a mean of 20 or more (table s1 ). no proteins found only in prok treated virions had a mean spectral count of more than 20, indicating that these proteins are present in the virions in low abundance. as discussed in the previous section, proteins not associated with the interior of the virion, including proteins embedded in the host derived viral envelope, can be identified by their absence in prok treated samples or by a size shift upon prok treatment. here we wanted to examine the localization within the vsv virion of host proteins that did not display any of these patterns (i.e. proteins that do not appear to be associated with the viral envelope). five of the chosen proteins were found in both whole and prok treated virion samples and had similar spectral counts in both sample types (table s1 ). we also chose two proteins, proto-oncogene tyrosineprotein kinase yes (yes1) and protein tyrosine phosphatase type iva 2 (ptp4a2; also called prl2), detected in prok treated virions but not whole virions, as proteins found in the treated samples would also be expected to be found in the whole virions from which they were derived (fig. 2) . to identify proteins associated with vsv rnp complexes, we also isolated and analyzed viral rnp complexes from a separate preparation of purified vsv virions by treating them with detergent and salt to release the nucleocapsid from the envelope and m protein while maintaining its association with the l/p polymerase complex. because protein quantities were chosen so that the amount of n and p protein was approximately equal for all virion samples (fig. 5) , the host protein associated with whole virions that was retained in the treated samples could be estimated. based on this, proteins were determined to be associated with the viral rnp complex, associated primarily with the interior of the virion but not the rnp or associated primarily with the exterior of the virion. most proteins were barely detectable in the rnp complexes, suggesting they do not associate with the viral rnp or that the association was disrupted under the conditions used for rnp isolation. only for e3 ubiquitin-protein ligase itchy (itch) were substantial amounts of protein detected in rnp. the m protein of vsv, as well as other rhabdoviruses, contains a late domain (ldomain) including a proline rich ppxy motif known to bind ww domains found in a number of cellular proteins [62] . vsv m protein has been shown to bind the ww domain of nedd4 (also detected in this analysis) [63] , and can presumably also bind other hect domain-containing e3 ubiquitin ligases, including itch, as has been reported for other viruses with l-domains including ppxy motifs [64] . however, given that itch is present in rnp complexes at levels that nearly equal the other two sample types, while m protein is sharply reduced, it seems likely that itch may also interact with one of the rnp proteins (n, p or l). three of the tested proteins, yes1, casein kinase i isoform alpha (csnk1a1) and heat shock cognate 71 kda protein (hspa8; also known as hsc70), while barely detectable in rnp, were found at similar levels in whole and prok treated virions, indicated they are primarily associated with the interior of the virions. in contrast, levels of ptp4a2 and putative atp-dependent rna helicase pl10 (d1pas1; also known as ddx3y), dropped sharply in the prok and rnp samples indicating they are not primarily located in the interior of the virion. one protein, coiled-coil and c2 domain-containing protein 1a (cc2d1a), identified by ms could not be detected by wb, indicating either protein levels below the threshold of detection, a misidentification of the protein based on homology, or a false-positive identification. the latter seems the least likely as the protein was identified in both sample types, with at least 6 consecutive amino acids matched in manual inspection. of the 6 proteins that could be detected by western blot, only 4 were present at similar levels in whole and prok treated virions, despite similar spectral counts in both sample types and the absence of a size shift. therefore, while these characteristics may suggest a protein associated with the interior of the virion, localization should be independently confirmed. to obtain an overview of the types of proteins most commonly associated with purified vsv virions, an enrichment analysis was conducted using gene ontology terms. this analysis was done for all three go databases (biological process, cellular component and molecular function), first using all the host proteins found in our analysis then looking specifically at proteins associated with prok treated virions (fig. 6) , as this is the highest purity sample and excludes proteins found exclusively on the exterior of the virion. related enriched terms were then clustered together to give an overall enrichment score. the most highly enriched clusters, when looking at all the proteins, were vesicles and vesicle mediated transport, protein localization and nucleotide binding (fig. 6 ). these were also the most highly enriched clusters when looking specifically at proteins identified in prok treated virions. the cluster cell adhesion was also relatively highly enriched when looking at all proteins, but less so in prok treated virions as would be expected. enrichment of proteins involved in vesicles and vesicle mediated transport, protein localization and cytoskeletal organization is consistent with known features of vsv assembly and budding. more than one third (88/257) of the proteins identified are represented in at least one of these 4 clusters, indicating many virion-incorporated host proteins are likely involved in these functions. transport of the vsv nucleocapsid to the site of budding has been shown to be dependent on microtubules [65] and is an important step in vsv assembly. furthermore, as seen for many enveloped viruses (reviewed in [66] ), vsv budding appears to use the host proteins involved in multivesicular body (mvb) formation which are relocated from endosomal membranes to the plasma membrane in an m protein dependent manner (reviewed in [67] ). however, the budding of vsv and related viruses may still have some unique features. while the specific members of the mvb pathway utilized by vsv for its budding are not clear, it is known that, unlike what has been observed for many other viruses, tsg101 is not essential for the budding of vsv and rabies virus [68] , while contradictory reports exist regarding the importance of vps4a [68, 69] . in contrast, the host ubiquitinproteasome system does appear to be essential [63, 69] . the lipid composition of the vsv envelope is consistent with that of its host cell, although levels of cholesterol and sphingomyelin are elevated [48] , indicating that unlike some other viruses such as influenza virus and hiv-1 [70, 71, 72] , vsv does not bud through host membrane regions enriched in lipid rafts. instead, vsv g and m proteins appear to initially localize to separate microdomains of the host plasma membrane but then either merge or cluster together with each other and host protein containing microdomains at the site of virus budding [73] . therefore, vsv readily incorporates the membrane proteins of its host. in pseudotyping experiments using both mixed virus infections and expression of viral or host proteins from vsv recombinants, non-vsv proteins can make up a significant portion of the protein in the vsv envelope, with incorporation levels up to 31% of that seen for vsv g protein reported [74, 75] . consistent with these observations, 46% (118/257) of proteins identified in our study are associated with the cellular component go term ''plasma membrane'', suggesting they were acquired along with the envelope. the tendency of vsv to indiscriminately acquire relatively large numbers of host membrane proteins has the potential to be exploited to help fine tune therapeutic vectors through the choice of cell line used to generate the viruses. for example, growing oncolytic vsvs in a cell line naturally expressing or engineered to express high levels of complement control protein may improve efficacy by slowing the rate of clearance by the host immune system following administration. while many of the proteins identified in vsv virions appear to be associated with viral assembly, budding or the host-derived viral envelope, they may also have additional functions that affect virus replication. furthermore, proteins were also identified that do not figure 5 . confirmation of host protein incorporation in virion preparations. protein lysates from uninfected (bhk) and vsv infected (bhk+ vsv) bhk-21 cells were separated by sds-page along with total protein from whole virions, proteinase k (prok) treated virions, and viral ribonucleoprotein complexes (rnp). loading of the virion samples was confirmed by ponceau s staining of the membrane. the position of the viral glycoprotein (g), nucleocapsid protein (n), phosphoprotein (p), and matrix protein (m) is indicated. western blots using primary antibodies against coiled-coil and c2 domain-containing protein 1a (cc2d1a), protein tyrosine phosphatase type iva 2 (ptp4a2), putative atp-dependent rna helicase pl10 (d1pas1), proto-oncogene tyrosine-protein kinase yes (yes1), casein kinase i isoform alpha (csnk1a1), heat shock cognate 71 kda protein (hspa8) and e3 ubiquitin-protein ligase itchy (itch), were conducted as indicated to determine the presence of the host proteins. approximate molecular mass of the proteins is given on the left. doi:10.1371/journal.pone.0104688.g005 have known associations with these functions. our study provides a valuable inventory of virion-associated host proteins for further investigation into their potential roles in vsv replication cycle, pathogenesis, and immunoreactivity. table s1 cellular proteins identified in whole and prok treated vsv virions following 1-d sds-page and uplc-ms/ms. (xlsx) figure 6 . functional enrichment analysis. enrichment analysis and clustering based on gene ontology terms was conducted for (a) all proteins identified and (b) proteins identified in the proteinase k (prok) treated virions, as described in the materials in methods. the five clusters in each database with the highest enrichment score are depicted here. numbers above the bars indicate the number of identified proteins associated with each cluster. doi:10.1371/journal.pone.0104688.g006 nonsegmented negative-strand viruses as vaccine vectors recombinant rhabdoviruses: vectors for vaccine development and gene therapy vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer vesicular stomatitis virus as an oncolytic vector rhabdoviridae. in: in: knipe dm and plunder and stowaways: incorporation of cellular proteins by enveloped viruses analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a rna interference screen presence of host icam-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and cd4(+)-t-cell depletion in human lymphoid tissue, a major site of replication in vivo level of icam-1 surface expression on virus producer cells influences both the amount of virionbound host icam-1 and human immunodeficiency virus type 1 infectivity host-derived icam-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity intercellular adhesion molecule 1 promotes hiv-1 attachment but not fusion to target cells virionbound icam-1 and activated lfa-1: a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type 1-infected individuals independently of the disease status and phase interaction between virion-bound host intercellular adhesion molecule-1 and the high-affinity state of lymphocyte function-associated antigen-1 on target cells renders r5 and x4 isolates of human immunodeficiency virus type 1 more refractory to neutralization role of cellular adhesion molecules in hiv type 1 infection and their impact on virus neutralization contribution of virion icam-1 to human immunodeficiency virus infectivity and sensitivity to neutralization host cell-derived complement control proteins cd55 and cd59 are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/ lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) cd59 incorporation protects hepatitis c virus against complement-mediated destruction human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored cd55 and cd59 and integral membrane cd46 at levels that protect from complement-mediated destruction a high-affinity inhibitor of human cd59 enhances complement-mediated virolysis of hiv-1: implications for treatment of hiv-1/aids extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope the paramyxoviruses simian virus 5 and mumps virus recruit host cell cd46 to evade complement-mediated neutralization vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions protein composition of the vaccinia virus mature virion identification of proteins associated with murine gammaherpesvirus 68 virions proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1 proteins of purified epstein-barr virus identification of proteins associated with murine cytomegalovirus virions comprehensive characterization of extracellular herpes simplex virus type 1 virions identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome virion proteins of kaposi's sarcomaassociated herpesvirus proteomic characterization of pseudorabies virus extracellular virions cellular proteins in influenza virus particles proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 proteomic analysis of purified coronavirus infectious bronchitis virus particles proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations distinct host cell proteins incorporated by siv replicating in cd4+ t cells from natural disease resistant versus non-natural disease susceptible hosts protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus 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vitro systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling empirical statistical model to estimate the accuracy of peptide identifications made by ms/ms and database search code developments to improve the efficiency of automated ms/ms spectra interpretation mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases future prospects for the analysis of complex biological systems using micro-column liquid chromatography-electrospray tandem mass spectrometry systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists biophysical studies of vesicular stomatitis virus structural domains of vesicular stomatitis virus. a study by differential scanning calorimetry, thermal gel analysis, and thermal electron microscopy the shape of vesicular stomatitis virus cytoskeletal proteins inside human immunodeficiency virus type 1 virions role of spectral counting in quantitative proteomics a proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with ww domains of cellular proteins: implications for viral budding rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells mechanisms for enveloped virus budding: can some viruses do without an escrt? rhabdovirus assembly and budding budding of ppxy-containing rhabdoviruses is not dependent on host proteins tgs101 and vps4a ubiquitin depletion and dominantnegative vps4 inhibit rhabdovirus budding without affecting alphavirus budding influenza virus assembly and budding in raft-derived microdomains: a quantitative analysis of the surface distribution of ha, na and m2 proteins evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides plasma membrane microdomains containing vesicular stomatitis virus m protein are separate from microdomains containing g protein and nucleocapsids foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles role of heterologous and homologous glycoproteins in phenotypic mixing between sendai virus and vesicular stomatitis virus the authors thank dr. sue moyer (university of florida college of medicine) for providing vsv reagents for this project and eric hastie for critical comments on the manuscript. key: cord-346777-zmmnn9b2 authors: lester, sandra; harcourt, jennifer; whitt, michael; al-abdely, hail m.; midgley, claire m.; alkhamis, abdulrahim m.; aziz jokhdar, hani a.; assiri, abdullah m.; tamin, azaibi; thornburg, natalie title: middle east respiratory coronavirus (mers-cov) spike (s) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies date: 2019-09-11 journal: access microbiol doi: 10.1099/acmi.0.000057 sha: doc_id: 346777 cord_uid: zmmnn9b2 middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic coronavirus that was identified in 2012. mers-cov infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. the mers-cov spike (s) protein is a major target for neutralizing antibodies in infected patients. the mers-cov microneutralization test (mnt) is the gold standard method for demonstrating prior infection. however, this method requires the use of live mers-cov in biosafety level 3 (bsl-3) containment. the present work describes the generation and validation of s protein-bearing vesicular stomatitis virus (vsv) pseudotype particles (vsv-mers-cov-s) in which the vsv glycoprotein g gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect mers-cov neutralizing antibodies under bsl-2 conditions. using a panel of human sera from confirmed mers-cov patients, the vsv-mers-cov particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live mers-cov. the results suggest that the vsv-mers-cov-s pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect mers-cov neutralizing antibodies in human sera. middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that is associated with respiratory virus infections ranging in severity from asymptomatic to severe respiratory illnesses and death. mers-cov was first isolated from a 60-year-old patient who died with viral pneumonia and acute renal failure in 2012 in saudi arabia [1, 2] . mers-cov cases have been identified in 27 countries, although all cases outside of the arabian peninsula have been associated with exportations from the arabian peninsula [3] [4] [5] [6] [7] . as of 28 february 2019, the world health organization (who) has reported 2374 confirmed cases of mers-cov infection globally, resulting in 823 deaths (https://www. who. int/ emergencies/ mers-cov/ en/). genetically, mers-cov most closely resembles severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus known as the first coronavirus to cause severe respiratory infections in humans. sars-cov emerged from an animal host in 2002 and caused a worldwide outbreak comprising an estimated 8000 cases with 800 deaths [7] [8] [9] [10] . similar to sars-cov, mers-cov infections represent zoonotic transmission events [11] [12] [13] . several sero-epidemiology studies have demonstrated that dromedary camels from the arabian peninsula and north africa have high titres of neutralizing antibodies against mers-cov, which can be detected in camel sera collected over 30 years [14] [15] [16] [17] . in contrast to the sars outbreak, which was brief, mers-cov cases continue to occur 7 years after its identification. the continuous spread of mers-cov serves as a constant reminder of the propensity of novel human coronaviruses to emerge from zoonotic reservoirs and cause severe disease in humans, and so continuous serological surveillance efforts remain essential for these viruses. the risk factors for camel-human transmission, humanhuman transmission and hospital outbreaks, and the roles of asymptomatic cases in human-human transmission are not fully understood. the clinical symptoms for mers patients overlap with those for other lower respiratory tract infections, further demonstrating the need to develop low biosafety-level laboratory-based diagnostic assays with high specificity and sensitivity. furthermore, countermeasures for the treatment or prevention of mers-cov infection, including vaccines, are unavailable. therefore, methods for measuring the potency and breadth of neutralization antibodies against mers-cov are essential to address these gaps in our knowledge, as well as to strengthen disease surveillance and disease preparedness platforms. the spike (s) protein of mers-cov is a surface glycoprotein that facilitates receptor binding, membrane fusion and viral entry into host cells via attachment to the cellular receptor dipeptidyl peptidase iv (dpp4), thus playing a pivotal role in mers-cov infection of the host cell [18] . the s protein is a major immunogenic component of covs and is the major target for neutralizing antibodies [19] [20] [21] [22] . laboratory tests, such as immunofluorescence assays (ifas) and conventional enzyme-linked immunosorbent assays (elisas), are commonly used screening tools for detecting anti-mers-cov serum antibodies. however, these assays often yield false-positive reactions due to cross-reactivity with other common human coronaviruses, and so confirmatory tests such as neutralization tests are utilized [23] . the most widely used confirmatory serological assays to detect and measure neutralizing antibody responses to mers-cov utilize neutralization of live virus, through either the plaque reduction neutralization test (prnt) or the microneutralization test (mnt) methods. however, due to the highly pathogenic nature of mers-cov and the absence of treatments and vaccines for mers-cov infection, prnt and mnt tests must be conducted under strict bio-containment procedures in a biosafety level 3 (bsl-3) laboratory. this limits epidemiological and pathogenesis studies globally. the development of an alternative method that can be reliably and conveniently used to determine the prevalence of mers-cov antibodies at a population level is crucial to prevent the spread of mers-cov. a safe and convenient way to detect neutralizing antibodies against mers-cov in serum is the use of viral pseudotypes. viral pseudotype particles have proven to be very valuable tools for studying virus entry pathways, evaluating the efficacy of vaccines, serological surveillance and gene therapy studies [24] [25] [26] [27] [28] [29] [30] [31] . several reports have utilized mers-lentivirus pseudotyped viruses in sero-epidemiological studies and basic research investigations [21, [32] [33] [34] [35] [36] . additionally, vesicular stomatitis virus (vsv) pseudotypes with mers-cov spike glycoproteins have been utilized to investigate the role of the mers s protein in virus-receptor-mediated entry, virus/host tropism and drug screening [37] [38] [39] . however, systematic comparative equivalency analysis between vsv pseudotypes bearing mers-cov spike glycoproteins and the conventional mnt using human sera from confirmed mers-cov patients have not been performed. the purpose of this study was to develop a vsv pseudotypebased neutralization assay to detect mers-cov s protein neutralizing antibodies and calculate its equivalence to traditional neutralization assays, therefore circumventing the use of live virus and the need for widely unavailable high-level containment biosafety facilities. here, we validate a mers-cov neutralization assay using a membraned vsv pseudotype system, where its glycoprotein is replaced with a luciferase reporter gene and its membrane is decorated with mers-cov s proteins. additionally, we perform equivalence testing in comparison with the conventional mnt. we demonstrate that these pseudoparticles are neutralized by sera from mers-cov-infected patients. furthermore, most of the patient sera samples have neutralization activities, and these results were consistent with the neutralization activity that was measured by the conventional microneutralization assay using live mers-cov. these results demonstrate that the mers-cov-s protein pseudotyped vsv particle-based neutralization assay would serve as a safe, reliable and highly specific alternative method to detect mers-cov neutralizing antibodies to be used for future sero-epidemiological studies. baby hamster kidney-21 (bhk-21) cells were cultured and maintained in dulbecco's modified essential medium (dmem) containing 5 % fetal bovine serum (fbs) (life technologies/gibco), and 1× penicillin/streptomycin (p/s) (sigma) at 37 °c under a 5 % co 2 atmosphere. vero (african green monkey kidney epithelial) cells were cultured and maintained in dmem containing 10 % fbs and 1× p/s 37 °c under a 5 % co 2 atmosphere. different vsv-based pseudotypes bearing either mers-cov-s or vsv-g glycoproteins were produced in bhk-21 cells. the pseudoparticle titrations and neutralization assays were carried out in vero cells. pooled normal human serum (pnhs) was purchased from lee biosolutions and used as a negative control serum. human serum from a single patient with laboratory-confirmed mers-cov infection was used as the positive control serum. the positive control serum was collected from the first imported case of mers-cov in the usa during the case investigation, with a neutralizing titre of 320. a laboratory-confirmed sars-cov patient serum sample and a panel of human sera with confirmed high neutralizing antibody titres to human coronaviruses 229e, hku1, oc43 and nl63 were used in this study to evaluate the vsv-mers-cov-s particle-based neutralization assay for potential cross-neutralization. a total of 52 human sera samples from mers-cov-infected patients in saudi arabia were used to examine equivalences. anti-vsv-g monoclonal antibody was purchased from kerafast, inc. a codon-optimized s gene from the mers-cov florida isolate (genbank accession number: kj829365.1) was synthesized by genescript and sub-cloned into pcaggs 2.0 eukaryotic expression vector. vsv-mers-cov-s pseudoparticles were generated as previously described [24] . the pseudoparticle titres were determined in vero cells. vero cells were seeded in 96-well black and white tissue culture-treated plates (perkin-elmer) and were inoculated with 50 µl of pseudoparticles five fold serially diluted in serum-free dmem (1× p/s in triplicate in . after 1 h adsorption, the inocula were removed, replaced with fresh dmem containing 10 % fbs and 1× p/s, and incubated at 37 °c. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit (promega, inc.) according to the manufacturer's instructions. the titre of the vsv-mers-cov-s pseudoparticles was defined as the highest dilution yielding 10 5 relative luciferase units (rlu). for the titration of vsv-mers-cov-s pseudoparticles, the pcaggs plasmids encoding vsv recombinant g and the empty vector (ev) pcaggs-ev were used to generate vsv-g pseudoparticles and pseudoparticles without heterologous protein (ev) as positive and negative controls, respectively. ten fold serial dilutions of vsv pseudotyped with mers-s (vsv-mers-s) or negative control pcaggs empty vector containing no glycoprotein (vsv-ev) were used to infect vero cells in 96-well plates. rlu were measured 24 h post-infection. the results are expressed as average rlu±standard deviation (sd) of triplicate wells from an experiment repeated three times with similar results. the error bars indicate the sd. briefly, vero cells were seeded in 96-well black and white tissue culture-treated plates 1 day prior to infection. human sera were heat-inactivated at 56 °c for 30 min, and diluted as two fold serial dilutions with an initial dilution of 1 : 40 in serum-free dmem containing 1× p/s. two hundred microlitres of each serum dilution were mixed thoroughly with 10 5 rlu-equivalent vsv-mers-cov-s pseudoparticles diluted to 200 µl and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. positive and negative control sera were included with each assay run. in triplicates, 100 μl of the pseudoparticleserum mixtures were transferred onto vero cell monolayers and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. after adsorption, 100 µl of dmem containing 10 % fbs p/s was added and incubated at 37 °c and 5 % co 2 for 23 h. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit according to the manufacturer's instructions. the results are expressed as the percentage neutralization ±sd of three parallel wells from three independent experiments. to calculate neutralization, the luciferase activity from each serial diluted human sera sample was normalized to that from the negative control, pnhs. neutralization was calculated as: (average rlu value of pnhs−average rlu value with test serum/average rlu value of pnhs)×100. reciprocal endpoint titres were defined as the highest dilution of the test serum sample that decreased rlu values by 80 % or more. serum samples that failed to show at least an 80 % reduction at the 1 : 40 dilution were considered to be below the limit of detection. the slides were first coated with poly-l-lys and seeded with hek-293t cells 24 h prior to transfection. the cells were transfected with 1 µg of pcaggs-mers-cov-s plasmid dna or mock transfection. at 24 h post-transfection, the cells' expression of mers-cov-s was detected by convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera; 1 : 400), followed by fitc-labelled anti-human iga/igg/igm (1 : 500) (abcam). dapi stain was used and cells were imaged using a zeiss axioimager microscope at 20× magnification. all graphs were generated with the graphpad prism 7 software. all results were calculated and presented as the means±sd obtained from duplicate or triplicate independent experiments. pearson's correlation analysis and the bland-altman method were used to assess the comparative analyses between the vsv-mers-cov-s pseudoparticle neutralization assay and the mnt assay. statistical analyses were performed using graphpad prism 7 software. the specimens used in this study were determined to be nonhuman subject research by the centers for disease control and prevention (cdc) institutional review board (irb). to establish vsv-mers-cov-s pseudotype neutralization assays, we generated vsv-mers-cov pseudoparticles with surface s protein and a firefly luciferase reporter gene as previously described [24] . the expression of mers-cov s proteins was confirmed by immunofluorescence at 24 h post-transfection in hek293t cells (fig. 1a) . it is known that bhk-21 cells do not have the s-binding mers-cov receptor, dpp4, and are therefore not permissible to mers-cov infection, but vero cells do [18, 39] . efficient packaging of mers-cov s on the surface of the vsv pseudoparticles and the ability of the pseudoparticles to enter target cells through receptor binding were confirmed by comparing the titrated inoculation of vero and bhk-21 cells of negative control vsv pseudoparticles containing no viral glycoproteins on their surface (vsv-ev) with that for vsv pseudotyped with mers-cov-s proteins, designated vsv-mers-s (fig. 1b and c) . as expected, in the vero cells the luciferase activity was proportional to the dilution of the mers-s pseudoparticle inoculum (fig. 1) . in comparison to the control pseudoparticles, the mers-s pseudoparticles demonstrated an approximately six fold increase in luciferase activity (fig. 1) . neither the mers-s pseudoparticles nor the control pseudoparticles induced high levels of luciferase activity in bhk-21 cells (fig. 1) , further supporting the notion that bhk-21 cells are resistant to mers-cov infection [39] . based on the rlu values observed at each serial dilution, it was decided to use a 1 : 200 dilution as the input pseudoparticle dose for further experiments for the mers-cov s protein pseudotyped vsv particle-based neutralization assay. to establish optimal parameters for the mers-cov s protein pseudotyped vsv particle-based neutralization assay, the cell density was optimized for maximum luciferase activity. no significant differences in luciferase activities were observed at 10 3 -10 5 cellsper well, although maximum luciferase activity was reached at 10 4 cells per well, and so 10 4 cells per well was selected as the standard assay cell density (fig. s1a , available in the online version of this article). next, we investigated the incubation time for optimal luciferase activity. the maximum rlu values peaked at 24 h post-infection and the rlu values decreased with prolonged incubation times (fig. s1b ). therefore, 24 h post-infection was determined to be the most suitable infection time for mers-cov-s pseudoparticles. to investigate whether anti-mers-cov human sera neutralized vsv-mers-cov-s pseudoparticles, vsv-mers-cov-s pseudoparticles were preincubated with two fold serial dilutions of pnhs serum (negative control), convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera) or anti-vsv-g monoclonal antibody. luciferase activity was reduced when vsv-mers-cov-s was preincubated with positive control serum in a dose-dependent manner (fig. 2a) . in contrast, preincubation with the negative control pnhs or anti-vsv-g monoclonal antibody did not significantly reduce luciferase activity at any dilution factor, thus demonstrating that vsv-mers-cov-s pseudoparticles were specifically neutralized by serum from an individual with a confirmed prior mers-cov infection (fig. 2a) . as expected, at any dilution factor, preincubation with anti-vsv-g monoclonal antibody efficiently neutralized vsv-δg pseudoparticles packaged with vsv-g, although neither the mers-cov positive control serum or the negative control pnhs neutralized vsv-δg pseudoparticles packaged with vsv-g (fig. 2b) . neutralization may be assessed as the percentage reduction in infectivity at a single dilution. since our goal is to measure the endpoint neutralization titres of human sera that possibly contain mers-cov neutralizing antibodies, we evaluated the percentage neutralization reduction curve of vsv-mers-cov-s pseudoparticles when preincubated with a mers-cov immune serum with a known live-virus endpoint neutralization titre. the percentage reduction in rlu (% neutralization) was determined by calculating the difference between the average rlu number in triplicate wells with the anti-mers-cov serum and the average rlu number in triplicate wells with the negative control pnhs sample, dividing it by the average rlu number in triplicate wells with the negative control pnhs sample and then multiplying the result by 100. neutralization of vsv-mers-cov-s pseudoparticles was dependent on the serum dilution, reducing rlu between 99 and 53 % at the dilutions tested (fig. 3) . using a target value of 80 % reduction in rlu in comparison to the pnhs control was within the linear range of reduction. our standard positive control mers-cov immune serum crossed the 80 % reduction line between 320 and 640 dilutions. this same serum has a known endpoint titre of approximately 320 in neutralization assays using live virus (unpublished data), demonstrating relative equivalence in the two assays for this serum. we previously measured the endpoint neutralizing antibody titres of 52 human sera collected from mers-cov patients using a live virus mnt. to determine if the vsv-mers-cov-s pseudoparticle neutralization test results in equivalent endpoint titres in comparison to the live virus mnt, we compared the endpoint titres from the live virus mnt to the endpoint titre necessary to achieve an 80 % reduction in rlu with vsv-mers-cov-s pseudoparticles. neutralizing antibody titres ranging from 40 to 1280 from 52 human serum samples against mers-cov were successfully detected by both neutralization assays (fig. 4) . sixty-seven per cent (n=35) of the serum samples had the same endpoint neutralization titres as those measured by either technique. we observed that approximately 29 % (n=15) of the serum samples gave higher neutralizing titres in the vsv-mers-cov-s pseudoparticle neutralization assay than the conventional mnt. furthermore, five sera with neutralization titres below the limit of detection by mnt had neutralizing titres that were detectable by the vsv-mers-cov-s pseudoparticle neutralization assay, thus demonstrating that the vsv-mers-cov-s pseudoparticle neutralization assay may be more sensitive (fig. 4) . the titres from both assays were then analysed statistically. pearson's correlation analysis of all data showed a strong correlation between the two neutralization assays, with r=0.9348, p<0.0001, 95 % confidence interval (ci)=0.8886 to 0.9622 (fig. 4a) . bland-altman method comparison analysis further demonstrated that these two methods are highly comparable. the average of the logarithmic difference of the two methods was 0.3269 (fig. 4b) . collectively, these data indicate that the vsv-mers-cov-s pseudoparticle neutralization assay can be a reliable alternative to the live virus-based mnt. there are six known human coronaviruses that cause respiratory disease in humans. in order to test the specificity of our vsv-mers-cov-s pseudoparticles and their potential cross-reactivity with other common human coronaviruses, we tested 40 serum samples from humans with no known anti-mers-cov exposure. when using the 80 % reduction in rlu as the cut-off value, none of the serum samples neutralized vsv-mers-cov-s pseudoparticles at any dilution factor (fig. s2) . we also tested potential cross-neutralization of vsv-mers-cov pseudoparticles with sera from other humans with confirmed infections with the human coronaviruses 229e, hku1, oc43, nl63 and sars-cov. using the neutralization parameters of 80 % or greater reduction in rlu, we found no cross-neutralization with 229e, hku1, oc43, nl63 or sars-cov anti-sera (fig. 5) . these data confirm the specificity of the vsv-mers-cov-s pseudoparticles. mers-cov continues to circulate on the arabian peninsula and can cause severe infections, including fatalities. at present, there are no antiviral therapeutics or approved vaccines to combat mers-cov infections. the presence of serum neutralizing antibodies ≥20 is a reliable indicator of prior mers-cov exposure. neutralization assays using live viruses are recognized as the gold standard serological assays to confirm the antibody responses to mers-cov infection. however, one major obstacle to performing the mnt assay is the requirement for access to high-level biocontainment laboratories, due to the use of live mers-cov. thus, only a limited number of facilities and researchers are able to perform such neutralization assays. additionally, determining the neutralization of infectious virus in this assay requires the assessment of cytopathic effect in 96-well plates, which is labour-intensive and time-consuming. furthermore, because of the large viral genome, reverse genetics for mers-cov is challenging, and still needs to be used under bsl-3 containment, thereby limiting molecular manipulation and studies. these limitations highlight the need to develop alternative methods for detecting mers-cov neutralizing antibodies. an alternative approach to overcome these shortcomings is the use of viral pseudotypes. generally, lentiviral and vsvbased pseudotypes are used for the study of enveloped viruses. several studies have utilized lentiviral and vsv pseudotype systems harbouring mers-cov spike glycoproteins to study viral entry, viral tropism and other basic science questions [27, 29, [37] [38] [39] [40] . in addition to being utilized to probe basic science questions, pseudoparticle-based neutralization assays have been used for serological surveillance of reservoir species for several other zoonotic viruses in addition to mers-cov [17, 33, [41] [42] [43] [44] [45] [46] . studies have employed lentiviral and env-deficient hiv-1 backbone vector pseudotype systems for mers-cov seroepidemiological surveillance in humans [32] [33] [34] [35] [36] . using dromedary camel sera, hemida et al. demonstrated that lentiviral-based mers-cov s pseudoparticles detected antibodies specific to mers-cov. in our hands, lentiviral particles grew to lower titres than vsv particles, making it challenging to prepare high volumes of stock for use in multiple studies. standardizing many particles for use in multiple studies is important for making comparisons between studies in this context. barlan et al. utilized vsv pseudotyped with mers-cov-s and demonstrated that susceptibility to mers-cov infection is enhanced by cellular proteases that cleave the s protein [37] . fukuma et al. demonstrated that infection with vsv-based pseudoparticles bearing truncated c-terminal mers-cov-s protein and non-truncated mers-cov-s protein was inhibited by rabbit anti-mers-cov s serum and that infection was dependent on the expression of dpp4 [40] . this demonstrated that vsv-based mers-s pseudoparticles can be utilized for neutralization assays. nonetheless, studies featuring seroepidemiological surveillance in humans utilizing a vsv pseudotype system bearing mers-cov spike glycoproteins had not previously been performed. this report describes the first comparison between the neutralization assay using vsv-mers-cov-s pseudoparticles and the mnt using a panel of human sera. this study is limited by only utilizing 52 specimens, which limits its statistical power. although analysing more specimens might yield a more powerful comparative analysis, the complexities of limited availability, diplomatic issues and biosafety concerns make acquiring mers-cov human specimens less feasible. despite this limitation, statistical analyses comparing mnt and vsv-mer-cov pseudoparticle neutralization indicate there was a positive correlation between the two methods. although there was a strong correlation between the two methods, the vsv-mers-cov-s pseudoparticle neutralization assay is slightly more sensitive than the conventional mnt. one potential explanation for this is the surface density of s protein. the generation of pseudoparticles by transient transfection in continuous cell lines, rather than producer cells that are more physiologically relevant, may lead to the production of large amounts of virus with less s protein on its surface. thus, the pseudoparticles may be more susceptible to neutralization because fewer neutralizing antibodies are required to block the entry of mers-cov. however, in corroboration with our data, fukushi et al. previously demonstrated that a neutralization test based on a vsv-based sars-cov-s protein bearing pseudoparticles was also more sensitive than the conventional virus neutralization test using live sars-cov [47] . in comparison with the mnt method, the pseudoparticle neutralization assay required half as much time to test for mers-cov neutralizing antibodies. more importantly, this assay can be performed in a low-biosafety containment laboratory. in addition to being faster and more sensitive than conventional neutralization assays while maintaining specificity, our vsv-mers-cov-s pseudoparticle system offers a convenient approach for examining the antigenic and neutralizing epitope differences among different s proteins from divergent strains of mers-cov. the data we have presented here use one s plasmid, but the pseudoparticles can be packaged using the spike sequence from other virus strains. because s protein is provided in trans in a pcaggs vector, it can be easily mutated or swapped for any spike protein an investigator is interested in studying. there are existing reverse genetics systems for human coronaviruses, including mers-cov, but those systems are more difficult to generate and require bsl-3 facilities [48] [49] [50] . using those full-virus reverse genetics systems are essential for viral replication and pathogenesis studies, but for neutralization studies, the presence of spike alone is sufficient. mers convalescent sera did not neutralize vsv particles packaged with vsv-g, demonstrating that neutralization was due to serum antibodies binding specifically to mers s. one of the main concerns in designing a mers-cov neutralization test is the presence of five other known human coronaviruses, four of which cause common infections. it had previously been shown that anti-coronavirus sera crossneutralize amongst similar coronaviruses [51] . in this study we determined that vsv-mers-cov-s pseudoparticles were not neutralized by antisera from alpha-or betacoronaviruses, further demonstrating the assay's high specificity. in conclusion, we established a neutralization test using a vsv-based pseudotyped virus possessing mers-cov s protein and expressing a luciferase reporter gene. we used the vsv-mers-cov-s pseudoparticle neutralization assay at bsl-2 containment, improving the safety of mers-cov neutralization assays. additionally, we found the system to be more rapid and sensitive and easier to use than a conventional microneutralization assay. these characteristics make the vsv-mers-cov-s pseudoparticle neutralization assay a more attractive, convenient and suitable assay for mers-cov screening and confirmatory serology testing. it offers a safe, rapid alternative method to monitor the potency and breadth of neutralization antibodies against mers-cov exposure in future seroepidemiological studies. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome emerging diseases. soaring mers cases in saudi arabia raise alarms the global spread of middle east respiratory syndrome: an analysis fusing traditional epidemiological tracing and molecular phylodynamics an outbreak of middle east respiratory syndrome 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evaluation of candidate vaccine approaches for mers-cov identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism adaptive evolution of mers-cov to species variation in dpp4 inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein development of a neutralization assay for nipah virus using pseudotype particles the production and development of h7 influenza virus pseudotypes for the study of humoral responses against avian viruses comparative serological assays for the study of h5 and h7 avian influenza viruses safe pseudovirusbased assay for neutralization antibodies against influenza a(h7n9) virus bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to sars-cov reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing red-mediated recombination cloning cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests the authors wish to acknowledge financial support from cdc lassi 2016. the authors declare that there are no conflicts of interest. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the centers for disease control and prevention or the us department of health and human services. the specimens used in this study were determined to be non-human subject research by the centers for disease control and prevention (cdc) institutional review board (irb). key: cord-276009-p98wjtjb authors: iyer, arun v.; pahar, bapi; boudreaux, marc j.; wakamatsu, nobuko; roy, alma f.; chouljenko, vladimir n.; baghian, abolghasem; apetrei, cristian; marx, preston a.; kousoulas, konstantin g. title: recombinant vesicular stomatitis virus-based west nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west nile virus strain lsu-ar01 date: 2009-02-05 journal: vaccine doi: 10.1016/j.vaccine.2008.11.087 sha: doc_id: 276009 cord_uid: p98wjtjb vesicular stomatitis virus (vsv) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. we constructed recombinant vsvs specifying either the indiana or chandipura virus g glycoprotein and expressing the west nile virus (wnv) envelope (e) glycoprotein. mice were intranasally vaccinated using a prime (indiana)-boost (chandipura) immunization approach and challenged with the virulent wnv-lsu-ar01. ninety-percent (9 of 10) of the vaccinated mice survived as compared to 10% of the mock-vaccinated mice after wnv lethal challenge. histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against wnv. extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of cd4(+) cd154(+) ifnγ(+) t cells in vaccinated, but not mock-vaccinated mice. similarly, vaccinated mice developed robust e-glycoprotein-specific cd8(+) t cell immune responses as evidenced by the presence of a high percentage of cd8(+) cd62l(low) ifnγ(+) cells. in addition, a sizeable population of cd8(+) cd69(+) cells was detected indicating e-specific activation of mature t cells and cd4(+) cd25(+) cd127(low) t regulatory (t reg) cells were down-regulated. these results suggest that vsv-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against wnv infections. west nile virus (wnv) was first isolated more than 70 years ago from a febrile patient in the west nile province of uganda [1] . wnv is a positive-sense rna virus belonging to genus flavivirus in the falviviridae family [2] . the lipid-bilayer membrane of the nascent virus contains 180 molecules of the envelope (e) and premembrane (prem) proteins organized into 60 asymmetric non-neurological clinical manifestations include rhabdomyolysis [22, 23] , pancreatitis [24] , hepatitis [25] , myositis, orchitis [26] , chorioretinitis [27] and cardiac dysrhythmias [28] . typically, less than 1% of patients suffer from west nile neuroinvasive disease (wnd) including west nile meningitis (wnm), encephalitis (wne) and acute flaccid paralysis (poliomyelitis-like syndrome, wnp) [29] . among wnd cases, an estimated 55-60% of the patients had wne resulting in an estimated 20% case fatality. additionally, 10-50% of mortalities in humans could be attributed to wnp [29] . the absence of effective treatment against wnv infection has encouraged vaccine development. a variety of different approaches have been employed to produce wnv vaccines including inactivated virus, subunit and dna-based vaccines. most of these vaccines appeared to be highly immunogenic, and in some cases protected against wnv-infection in experimental animals [30] . recently, recombinant viruses expressing wnv antigens have been shown to induce strong immune responses and protection against wnv challenge in animals. specifically, a recombinant live canarypox-vectored vaccine expressing the prem protein and the e glycoprotein induced strong immune responses in horses and cats [31] [32] [33] [34] , that appeared to be partially protective [35] . other viral-vectored vaccines that elicited protective immune responses in mice include a lentivirus vector based vaccine (trip/se wnv ) [36] , and a measles virus-vectored vaccine [37] . recombinant yellow fever virus (yfv) has also been used to express wnv prem and e proteins based on the extensive safety record of the yfv attenuated vaccine [38, 39] . a yfv recombinant vaccine (chimerivax tm ) has shown good immune responses in hamster, mice, non-human primates and humans [40] [41] [42] . a phase ii clinical trial with chimerivax tm -wnv is currently underway [43] . vsv is an enveloped, negative strand rna virus belonging to the rhabdoviridae family. natural vsv infections of humans are rare causing at most mild flu-like illness [44] . vsv infectious viruses can be efficiently recovered by a reverse genetic approach that utilizes multiple plasmids expressing vsv genes. this methodology has enabled the rapid construction of recombinant vsv viruses expressing a variety of viral and bacterial antigens for vaccine purposes including influenza virus, bovine diarrhea virus, cotton-tail papillomavirus, human immunodeficiency virus, simian immunodeficiency virus, respiratory syncytial virus, hepatitis c, measles virus, ebola virus, lassa fever virus, marburg virus, severe acute respiratory syndrome virus (sars), and herpes simplex type-2 virus [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] . recombinant vsvs have been also constructed and tested as vaccines for bacterial pathogens including mycobacterium tuberculosis and yersinia pestis [64, 65] . vsv-vectored vaccines have been administered via intranasal, intramuscular and subcutaneous routes and have been shown to elicit robust mucosal and systemic humoral and cellular immune responses [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [66] [67] [68] . we constructed recombinant vsvs expressing the wnv e glycoprotein. a prime-boost approach was employed utilizing two different recombinant vsvs expressing either the indiana or the chandipura g glycoproteins for priming and boosting immunizations, respectively. intranasal immunization of mice conferred high protection against lethal challenge with the virulent wnv strain wnv-lsu-ar01 [69] . neuronal necrosis was observed in mockvaccinated but not in vaccinated mice. these results suggest that vsv recombinant vaccines expressing the wnv e glycoprotein may be efficacious intranasal vaccines for animal and human use. baby hamster kidney cells (bhk-21) were obtained from the american tissue culture collection (atcc). these cells were grown using dulbecco's modified minimal essential media (dmem) supplemented with 10% fetal bovine serum (fbs) and appropriate amounts of antibiotics. the west nile virus envelope (e) gene was obtained by first producing a cdna of the e gene from the wnv-lsu-ar01 strain, and subsequently cloning this gene into the pcdna3.1 plasmid (invitrogen, inc.) after pcr amplification. the e gene was further amplified by pcr from this plasmid using primers that introduced unique noti and bamhi sites at the 5 and 3 using 5 wne-flag-not-i (5 -gacgacgcggccgc-atgtttaactgccttggaa tgagc-3 ) and 3 wne-flag-bamhi (5 -gcagcaggatccagcgtgcacgttcacgg agagg-3 ) primers. noti and bamhi sites are italicized. the fragment was then cloned into plasmid p3xflag-cmv-14 (sigma) placing the flag epitope coding sequence downstream and in-frame with the e glycoprotein sequence. all recombinant plasmids were confirmed by restriction endonuclease digestion and dna sequencing. bhk-21 cells were transfected with the wnv e-3xflag plasmid using lipofectamine 2000 (invitrogen) as suggested by the manufacturer. e glycoprotein was detected at 48 h post-transfection using anti-flag (sigma) and anti-west nile rabbit polyclonal antibody (abcam). for immunofluorescence assay (ifa), cells were washed twice with phosphate buffered saline (pbs) and fixed with ice-cold methanol. cells were then washed with pbs and wells were blocked with 2% bsa and 5% goat serum in tbs (tris-buffered saline) for 1 h. mouse anti-flag antibodies (sigma) in blocking buffer and rabbit anti-wnv antibodies were added to respective wells at a 1:500 dilution and incubated for 1 h at room temperature. cells were then washed six times with tbs and the secondary antibody alexa fluor ® 488 goat anti-mouse igg and goat anti-rabbit igg (invitrogen) were added to the respective wells at the same dilution. cells were incubated in dark for 1 h. finally, cells were washed six times with tbs and observed under a fluorescence microscope. plasmid clones that efficiently expressed the wnv e gene were used as the template for pcr amplification of the gene, while at the same time introducing unique xhoi and nhei sites at the 5 and 3 ends of the gene fragment using 5 -xn2-xho-i (5 -ccgcggctcgagatgttt aactgccttggaatgagc-3 ) and 3 -xn2-nhe-i (5 -gacgacgctagcggatcactac ttgtcatcgtc-3 ) primers. xhoi and nhei restriction sites are italicized. this dna fragment was cloned into the pvsv-xn2-in and pvsv-xn2-ch transfer vectors. cells were infected with recombinant vaccinia virus expressing t7 polymerase (vtf7-3) at a multiplicity of infection (moi) of 10 for 1 h. subsequently, bhk 21 cells were co-transfected with pbs-n, pbs-p, pbs-l and pvsv-xn2 containing the wnv e gene and recombinant virus was recovered as described in detail previously [70, 71] . control viruses having no exogenous inserted genes were also produced using pbs-n, pbs-p, pbs-l and the pvsv-xn2 (empty vector). anti-flag and anti-wnv-e antibodies were used to detect expression of the e glycoprotein by ifa in vsv-infected bhk cells. viral isolates expressing high amounts of the wnv e glycoprotein were selected through multiple rounds of plaque purification. viral titers were determined and stocks were stored at −80 • c for vaccination studies. all animal studies were carried out after the appropriate approvals were obtained from the lsu institutional animal care and use committee (iacuc) and bsl3 biosafety committee. four groups of ten 4-week-old female balb/c mice (harlan, in, usa) were used in this study. each individual mouse was identified with an ear tag (national band and tag company, ky, usa). group i (vaccine group): these animals were mildly anesthetized by inhalation of 2-3% isoflurane and 10 l dose of vaccine containing 10 5 pfu of the vaccine (rvsv-in-wnv e) was administered intranasally using a 10 l pipette (5 l per nostril). animals were boosted with the rvsv-ch-wnv e at 21 days post-vaccination using the same technique. one mouse from this group was not included in the fluorescenceactivated cell sorting (facs) analysis due to sample preparation problems (n = 9). group ii (control for vaccine group): control group animals were vaccinated in the same way as described above with the exception that they were inoculated with 10 l of uninfected cell culture supernatant. these animals were boosted at 21 days post-vaccination with uninfected cell culture supernatant. animals belonging to groups i and ii were humanely euthanized at 14 days post-boost. spleens were collected in eppendorf tubes containing rpmi and processed by flow cytometry for intracellular cytokines and cell-surface markers associate with memory t cells, regulatory t cells and cytotoxic t cells among others. for serology, animals were bled by the sub-mandibular route (cheek bleed) using golden-rod lancets (medipoint, ny). animals were bled on 21 days post-vaccination and 14 days post-boost. blood was collected in becton dickinson microtainers with serum separators (becton dickinson). these 20 animals were treated exactly in the same way as groups i and ii until the boost stage. at 10 days post-boost, these animals were transported to the animal biosafety level-3 (absl-3) facility for acclimatization. blood was collected at 14 days postboost (before challenge). animals were challenged intraperitonially with 10 5 pfu of wnv-lsu-ar01 and observed 2-3 times a day for 18 days. animals showing severe neurological symptoms (like ataxia and hunching posture) were humanely euthanized and dead animals were surgically processed immediately (thoracic and abdominal cavities opened up and placed in 10% formalin jars) for pathological studies. serum samples were inactivated by incubation at 65 • c for 30 min. serial two-fold dilutions of the serum were incubated with equal volumes of 50 pfu of wnv-lsu-ar01 at 37 • c for 1 h. serumvirus mixtures were then added to vero cell monolayers in 12-well plates in triplicates and the plates were incubated for another hour. plates were then overlaid with dulbecco's modified minimum essential media (dmem) containing 1% methyl cellulose and 2% fetal bovine serum. plates were incubated at 37 • c for 72 h and then fixed with 10% formalin in phosphate buffered saline (pbs). plates were washed three times with pbs and stained with 0.01% crystal violet. plaques were counted and the highest dilution of serum resulting in reduction of 90% of the plaques was noted. mouse splenocytes were adjusted to 10 7 cells/ml. one-hundred microlitre aliquots of splenocyte suspension was incubated with appropriately diluted concentrations of antibodies for 30 min at room temperature. cells were washed once with pbs and fixed with 1x bd stabilizing fixative buffer (bd biosciences) in distilled water. cells were kept protected from light at 4 • c and flow cytometric acquisition was completed within 24 h of staining. polychromatic (7 parameters) flow cytometric acquisition was performed on a lsr ii becton dickinson instrument having three lasers (488 nm blue laser, 633 nm red laser and 407 violet laser) by using fitc, pe-texas red, apc, apc-cy7 and pacific blue as the available fluorochrome parameters. single-stained controls for each fluorochrome were used for setting flow cytometry compensation. monoclonal antibodies including cd127 fitc (a7r34, ebioscience), cd62l pe-texas red (mel-14, invitrogen), cd25 apc (3c7, bd biosciences), cd4 apc-cy7 (gk1.5, bd biosciences) and cd8a pacific blue (53-6.7, bd biosciences) were used. at least 50,000 events were collected by gating on cd4 + t cells and those data were analyzed using flowjo software (treestar inc.) version 8.7.1. to test cd4 + or cd8 + t lymphocytes subsets for ifn␥ production, intracellular cytokine flow cytometry (cfc) assay was employed in response to each wnv peptide pool stimulation as described previously [72] . briefly, processed splenocytes were resuspended at 1 × 10 6 cells/ml in complete rpmi-10 with 10% fcs, and stimulated with 2 different wnv peptide pools at a final concentration of 1 g/ml of each peptide pool. peptide pools (15-19mers with 10-11 amino acids overlap) derived from the wnv e glycoprotein were based on the wnv-ny99 e amino acid sequence (nih biodefense and emerging infections research resources repository, niaid, nih). the 67 peptide array was divided to generate two peptide pools. peptide pool 1 (pp1) was made of peptides 1-34 and peptide pool 2 (pp2) was composed of peptides 35-67. for positive controls, pma (50 ng/ml, sigma) and ionomycin (1 g/ml, sigma) were used. negative controls had no antigen or mitogen stimulation. brefeldin a (10 g/ml, sigma) was added to cultures after the first hour, in a 6-h incubation period. following stimulation, cells were stained for cell-surface markers with directly conjugated mabs to cd69 fitc (h1.2f3, bd biosciences), cd62l pe-tr, cd4 apc-cy7 and cd8a pacific blue for 30 min at room temperature and washed with dpbs/bsa wash buffer. cells were then fixed and permeabilized by using cytofix/cytoperm (bd biosciences), washed twice in perm buffer (bd biosciences), and stained with intracellular mabs. ifn␥ pe (xmg1.2, bd biosciences) and/or cd154 apc (mr1, ebiosciences) were added to cells and incubated at room temperature for 30 min. single color and isotype-matched control antibodies were used to confirm staining specificity. after washing, cells were resuspended in 1% paraformaldehyde in pbs and stored in the dark at 4 • c. data were acquired within 24 h of staining using a lsr ii instrument (bd immunocytometry system) and facsdiva software (bd immunocytometry system). for each sample, 50,000 events were collected by gating on cd4 + t cells. data analysis was performed using flowjo software. gated cd4 + and cd8 + t cells were further analyzed for its cytokine production. positive cytokine responses were determined based on the percentage of cytokine responses obtained above background responses (unstimulated medium control) in each experiment. tissues (brain, lung, liver, bilateral kidneys, heart, spleen, skull, and vertebra) were collected from the mice, euthanized or after dead, and fixed by immersion in 10% neutral buffered formalin. the skull and vertebra were decalcified in 10% formic acid for 3 days. all sampled tissues were routinely processed into paraffin, and 3-4 m sections were cut for hematoxylin and eosin staining (h&e). h&e sections of the nasal olfactory epithelium and bulb in the skull and four sections of the spinal cord including two consecutive anterior cervico-thoracic and two consecutive lumbar-sacral posterior sections in the vertebrae were examined under the light microscope. graphical presentation and statistical analysis of the results were performed by two-tailed student's paired t-test using the graphpad prism 4.0 (graphpad software inc., sandiego, ca). expressions of cd62l, cd154, cd25 and cd127 between immunized and mock challenged mice were determined by nonparametric mann-whitney t-test. for all statistical analysis, results were considered significant if p < 0.05. mouse survival analysis was done with graphpad prism 5.01 (graphpad software inc., sandiego, ca) using the gehan-breslow-wilcoxin test. the wnv-lsu-ar01 strain was isolated from a dead blue jay (cyanocitta cristata) in louisiana in 2001. recently, the entire genome of this strain was sequenced and phylogenetically compared to 75 full wnv genomes deposited in genebank [69] . the e gene was amplified from viral rna using specific primers as described in section 2 and cloned into plasmid p3xflag (sigma) placing the entire open reading frame of wnv e in-frame with the 3xflag coding sequence resulting in the addition of the 3xflag amino acid sequence immediately after the last carboxyl terminal amino acid of the e glycoprotein. the p3xflag-e plasmid was transfected into baby hamster kidney cells (bhk-21) and e glycoprotein expression was detected at 48 h post-transfection using anti-flag monoclonal antibody. the anti-flag antibody detected e glycoprotein expression in 3xflag-e transfected bhk cells, while mock-transfected bhk cells failed to react with the anti-flag antibody. to construct recombinant vsvs expressing the e glycoprotein, the e gene was amplified with primers engineered to have unique xhoi and nhei restriction sites at the e 5 and 3 termini, respectively. the amplified e gene (with the 3xflag coding sequence) was cloned within the unique xhoi and nhei restriction sites of plasmids pvsv-xn2-in and pvsv-xn2-ch containing the indiana and chandipura g glycoprotein gene, respectively (fig. 1a) . recombinant vsv was recovered after co-transfection of pvsv-xn2-e with three other plasmids encoding the vsv polymerase subunits (p and l), and the nucleocapsid (n), purified by filtration and extensively plaque-purified. the appropriate insertion of the wnv gene within the vsv genomes was confirmed by direct dna sequencing of viral rna after rt-pcr amplification of specific cdna regions. wnv e expression was readily detected by indirect immunofluorescence assay (ifa) using anti-flag monoclonal antibody in recombinant vsv-infected bhk cells, while wnv e was not detected in mockinfected bhk cells (fig. 1b) . cell lysates from bhk-21 cells infected with recombinant vsvs expressing the wnv e glycoprotein were tested for e glycoprotein expression in western immunoblots. anti-flag antibody readily detected major protein species with apparent molecular masses of approximately 53-55 kda, respectively in agreement with previous reports (fig. 1c) [33, 73] . four groups of 4-week-old balb/c mice (harlan, in, usa) were used for the vaccine-challenge experiments. all four groups of mice were vaccinated by intranasal administration of 10 5 pfu of pvsv-xn2-in-e recombinant virus at day 0 and boosted with pvsv-xn2-ch-e (10 5 pfu) 21 days post-vaccination ( fig. 2a) . mice in groups i and ii were processed for immunological analyses (see section 2), while groups iii and iv were challenged with 10 5 pfu of wnv-lsu-ar01 administered intraperitoneally. mice in the challenge groups were observed for 18 days for clinical signs including ruffled fur, ataxia, hunching posture, lethargy and mortality. vsv-e vaccinated and boosted animals exhibited 90% survival, while only 10% of the mock-vaccinated animals survived wnv-lsu-ar01 challenge (p = 0.004) (fig. 2b) . vaccinated animals appeared to have mild clinical signs post-challenge including mild fur ruffling, but recovered quickly to a full healthy status. in contrast, mockvaccinated animals exhibited severe clinical signs post-challenge including high degree of fur ruffling, ataxia, lethargy and eventually death. post-mortem histopathological examination revealed that none of the vaccinated mice showed any central nervous system (cns) pathology as compared to mock-vaccinated animals, which exhibited severe neuronal necrosis and lymphoplasmacytic perivascular cuffing (fig. 3) . the single mouse in the vaccinated group that died at 12 days post-challenge had suppurative rhinitis which may be suggestive of bacterial infection. mild suppurative inflammation was also observed in the visceral pleura and subpleura of three mock-vaccinated mice that died before 11 days post-challenge (not shown). there were no significant histopatho-logical abnormalities within other tissues examined. in a separate set of experiments mice were vaccinated via the intramuscular route and challenged with a different strain of wnv (wnv-ny99) 9 weeks post-boost. the vaccine efficaciously protected 70% of the vaccinated mice (not shown). the ability of mouse sera to neutralize wnv-lsu-ar01 strain was tested in a standard plaque reduction neutralization test (prnt 90 ). vaccinated animals developed strong neutralizing antibody responses against the lsu-ar01 at 21 days after primary vaccination. specifically, 9 of 10 mice developed prnt 90 cd154 expression in cd4 + t cells is intimately involved in the polyclonal activation of immature b cells [74] . therefore, we compared the expression of cd154 in both vaccinated and mockvaccinated mice after in vitro stimulation with pma/ionomycin followed by facs analysis (see section 2). these experiments revealed the presence of a significantly higher population of cd4 + cd154 + ifn␥ + t cells in vaccinated mice compared to mockvaccinated mice (mean value 1.73% versus 1.0% in vaccinated and mock-vaccinated mice respectively, p < 0.0001, fig. 4a and c), as also indicated by the observed differences in their mean fluorescence intensities ( fig. 4b and d) . antigen-specific cytokine responses were determined in all vaccinated and mock-vaccinated mice. specifically, wnv-e specific t cell responses were measured using cytokine flow cytometry (cfc) to determine ifn␥ responses. overall, 7 of 9 vaccinated mice had detectable ifn␥ responses (ranged from 0.07 to 0.80%) in splenic cd8 + t cells. cd4 + t cell positive ifn␥ responses were absent in any of the vaccinated mice. both peptide pools 1(e amino acids 291-554) and 2 (e amino acids 544-791) appeared to contain t cell epitopes, however, peptide pool 1 contained dominant t cell epitopes. one of the 9 mice developed antigen-specific ifn␥ responses against both the wnv-e peptide pools. none of the mock-vaccinated mice had any detectable ifn␥ responses above background levels. cd62l is a lymphocyte homing marker that is generally associated with extravasation of activated t cells to peripheral sites of inflammation. generally, increased percentages of cd8 + t cells were present in the vaccinated mice compared to the mock-vaccinated mice (mean 18.3% and 15.3% for vaccinated and mock-vaccinated mice respectively, p = 0.01) (fig. 5a) . cd8 + t cell subsets in all vaccinated mice had lower cd62l expression compared to mockvaccinated mice (p = 0.0003) (fig. 5b) . to further characterize the cells responsible for inducing cytokine responses, antigen-specific cytokine positive cells were determined. a significant population (0.73%) of the ifn␥ positive cells was memory cells (cd8 + cd62l − ) (fig. 5c) . cd69 is an early activation marker indicative of the presence of antigen-specific stimulation of mature t cells [75] . cd69 up-regulation of activated cd8 + t cells was detected in all the vaccinated mice following antigen stimulation compared to mock-vaccinated mice (mean 1.8% versus 0.8% in vaccinated and mock-vaccinated mice, p = 0.012) indicating e-specific stimulation of mature t cells in vaccinated animals (fig. 5d) . initial determination of cd4 + t cell percentages in splenocytes revealed no significant differences between vaccinated and mock-vaccinated mice (fig. 6a) . cd127, the ␣-chain of the il7 receptor, in combination with cd25, the ␣-chain of the il2 receptor, were used to define the relative abundance of t reg cells within the population of conventional t cells [76] . analysis of cd4 + cd25 + cd127 low cells revealed that vaccinated mice had a significantly lower population of these cells (mean 6.3%) in comparison to the mock-vaccinated mice (mean 7.3%) (p < 0.05) (fig. 6b and c) . vsv-vectored vaccines have shown exceptional promise for protecting animals and humans against different viral and bacterial pathogens. a vsv-vectored vaccine expressing the wnv-e glycoprotein was constructed and found to efficiently protect mice after intranasal administration against lethal wnv challenge. the salient features of this vaccine study are: (1) a prime-boost intranasal vaccination approach with recombinant vsvs expressing the wnv e glycoprotein produced robust cd8 + ifn␥ + t cell responses; (2) this vaccine approach produced strong neutralizing titers against the wnv; (3) vaccinated mice were protected against lethal challenge and they were free of neuronal necrosis, while unvaccinated mice there was no statistically significant difference observed between these two groups. (b) representative dot plots showing the gating strategy for t reg cells derived from spleenocytes. cd4 + t cells were first gated and plotted for cd25 and cd127. cd4 + cd25 + cd127 low t cells were defined as t regs. (c) percentage of cd4 + cd25 + cd127 low t reg cells. a statistically significant difference (p < 0.05) was observed between wnv-vaccinated and mock-vaccinated mice. exhibited severe neuronal necrosis and inflammation in the brain. these results suggest that a prime-boost vsv-vectored intranasal vaccine approach induces strong humoral and cellular immune responses that protect mice against wnv-induced neuronal necrosis. mucosal surfaces constitute the natural route of vsv infections. vsv is primarily a veterinary viral pathogen that infects cattle, horses, sheep and other animals. vsv infects animals via transmucosal and transcutaneous routes [77] . vsv may also be transmitted through sandflies, blackflies and mosquitoes [44, 78] . the vsv g glycoprotein is a potent immunogen and also serves important functions in virus-entry and virus-induced cell fusion [79] . recombinant vsvs expressing a variety of viral and bacterial antigens have been constructed. vaccine studies with these recombinant vsvs have showed that intranasal and intramuscular administration of the rvsvs were safe and efficient in inducing protective humoral and cellular immune responses against a variety of pathogens [78] . of particular interest is the ability of the vsv-vector system to elicit strong humoral and cellular immune responses via the intranasal route [48, 49, 59, 60, 65, 80] that can be substantially easier to administer than intramuscularly injected vaccines. in these vaccine studies, although the "empty" vsv vector elicited robust humoral and cellular immune responses against vsv, these responses did not contribute to protection against a variety of pathogens indicating that specific immune responses against the expressed transgene were primarily responsible for protection [49, [58] [59] [60] 81, 82] . we constructed rvsvs that expressed the wnv-e glycoprotein and either the vsv indiana g glycoprotein, or the chandipura vesiculovirus g glycoprotein. this pair of rvsvs was used in a prime-boost-vaccination approach to maximize humoral immune responses against the wnv-e glycoprotein expressed by both viruses, while minimizing the anamnestic immune response against the vsv vector targeted predominantly against the g glycoprotein. this is largely accomplished because the chandipura g and the vsv-indiana g glycoproteins are approximately 60% different in their amino acid sequences [83] . recombinant vsvs are known to non-specifically incorporate certain other viral and cellular glycoproteins into their virions without adversely affecting viral infectivity [70] . the insertion of the foreign e gene into the vsv genome did not adversely affect viral replication and infectivity, because rvsv containing the e gene replicated to similar titers with those of the vsv control virus that did not have a foreign gene inserted within their genomes (not shown). moreover, rvsv-e isolates were stable, since multiple serial passages of virus stocks in bhk cells did not affect e glycoprotein expression and genomic stability (not shown). although it is unclear whether the wnv e glycoprotein is inserted into vsv envelopes, these results suggested that rvsv-e were stable retaining wild-type levels of viral replication and infectivity. recombinant vsv-e expressed wnv-e glycoprotein to high levels in bhk cells and the expressed e glycoprotein appeared to be fully glycosylated as evidenced by the apparent molecular mass of approximately 53-55 kda in sds-page in agreement with published reports [33, 73] . based on the known strong immune responses generated by vsv, especially when administered via the intranasal route, we devised an experimental vaccine protocol to vaccinate mice through the intranasal route using a prime-boost strategy. this prime-boost-vaccination approach resulted in 90% (9 of 10) of the mice surviving lethal challenge with the wnv-lsu-ar01 virulent strain. the single mouse from the vaccinated group of mice that died late in the experiment (12 days post-challenge) appeared to die from wnv-unrelated causes, since histopathological examination showed severe suppurative rhinitis but no histological abnormality in the brain. therefore, the rvsv-e prime-boost intranasal vaccination protocol was highly efficacious in protecting mice against wnv infection. similar results were obtained in a different experiment in which mice were vaccinated via the intramuscular route and challenged with wnv-ny99 strain instead of the lsu-ar01 9 weeks post-boost. in this experiment 70% of the mice survived indicating that intramuscular immunization may also provide protective immune responses against wnv infection. primary wnv infection is thought to result in local replication of the virus in peripheral organs and viremia that ultimately results in virus invading the cns. wnv mortality is thought to be largely caused by replication of the virus in the cns tissues of animals and the resultant immunopathological damage of cns tissues. accordingly, unvaccinated mice showed obvious clinical signs of neurological disease such as ataxia, hunching posture, lethargy and hindlimb paralysis. histopathological examination of brain tissues showed neuronal necrosis, perivascular cuffing, and microgliosis. in contrast, only a few vaccinated mice developed mild clinical signs such as mild ruffled fur, but recovered quickly. importantly, none of the vaccinated mice exhibited any neuronal necrosis. the interaction of cd40 on b cells with cd154 (cd40l) on cd4 + t cells results in t cell mediated activation of b cells resulting in immunoglobulin class switching, somatic hypermutation and proliferation [84] [85] [86] . accordingly, cd4 + cd154 + ifn␥ + t cells were up-regulated in vaccinated but not control mice indicating generation of t cell mediated b cell activation. the specificity of this response is not discernable, since it may be due to either or both vsv and wnv antigens. however, strong neutralizing antibody titers were also produced against wnv indicating the induction of especific humoral immune responses. this result is in agreement with previous reports showing that other vsv-vectored vaccines induced strong humoral immune responses against different vsvexpressed antigens. specifically, recombinant vsvs expressing either the respiratory syncytial virus f glycoprotein [49] , or rvsv expressing the severe acute respiratory syndrome (sars) corona virus (sars-cov) produced high antibody titers against the f glycoprotein and sarc-cov spike (s) glycoprotein, while strong immune responses against the vsv virus was noted [59] . the wnv e glycoprotein contains multiple predicted and experimentally verified cytotoxic t cell (ctl) epitopes [87] [88] [89] [90] . the availability of a library of overlapping peptides derived from the wnv e glycoprotein allowed the elucidation of antigen-specific cellular immune responses. peptide pool 1 composed of the first 34 peptides averaging 12-18 amino acids each generated stronger cellular cd8 + ifn␥ + t cell responses in in vitro proliferation assays, in comparison to peptide pool 2, which represented the carboxyl terminus-half of the wnv e glycoprotein. peptide pool 1 contains the experimentally verified ctl epitope rsycylat (e 347-354) while peptide pool 2 contains the ctl epitope ialtflav (e771-778), both of which have been shown to confer protection against lethal wnv-challenge in mice [87, 89] . in vitro stimulation of lymphocytes from vaccinated mice revealed the presence of antigen-specific ifn␥ responses specifically in cd8 + cd62l low t cells. cd62l (lselectin) mediates adhesion of resting lymphocytes to peripheral lymph nodes. typically, high expression of cd62l (cd62l hi ) reveals entrapment of lymphocytes within lymph nodes, while low cd62l (cd62l low ) cell-surface expression (the result of t cell activation) is indicative of lymphocyte extravasation to sites of inflammation [91] . splenocytes from vaccinated mice had significantly lower expression of the cd62l marker on e-specific ifn␥ + cd8 + t cells revealing activation and extravasation of these cells to peripheral sites, potentially involved in killing virus-infected cells prior to transmission to the cns. cd69 is an early activation marker that is absent in resting lymphocytes [75] . the up-regulation of the cd8 + cd69 + e-specific t cell responses in vaccinated versus mock-vaccinated mice provides additional evidence for the stimulation of t cells. accordingly, cd8 + cd69 + e-specific population of t cells was up-regulated in vaccinated versus mock-vaccinated mice indicating the generation of activated memory cd8 + t cells. it is unclear whether the observed cd8 + t cell memory responses confer long-term immunity against wnv infection. t regs are known to play important roles in down-regulation of anti-self immune responses [92] , and to suppress proliferation and cytokine production of effector t cells [93] . typically, during viral infections, up-regulation of humoral and cellular immune responses causes down-regulation of t reg activation. typically, t regs express the foxp3 and cd25 markers. the il-7 receptor cd127 marker expression is inversely correlated to foxp3 expression and cd127 low cd25 + cells have been shown to be positive for foxp3 [93, 94] . consequently, the cd25 + cd127 low population was used to define t regs. as expected, there was a negative correlation between the relative population of t reg cells (cd4 + cd25 + cd127 low ) and antigen-specific ctl responses in the vaccinated mice. however, the specificity of this immune response cannot be discerned, since it most likely is caused by both vsv and e glycoprotein antigens. a variety of experimental vaccine approaches have been reported to generate protective humoral and cellular immune responses against flaviviruses and specifically wnv. the relative role of humoral versus cellular immune responses has been extensively debated in the literature. certain studies have suggested that a strong humoral immune response evidenced by the production of high titer anti-wnv titers is necessary and sufficient to protect mice from cns infection, while other reports have argued that a cellular immune response characterized by a robust anti-wnv cd8 + t cell responses is necessary for protecting and clearing brain tissues from wnv [89, 95, 96] . one report has argued that ctl-immune responses may result in exacerbated immunopathology in brain and cns tissues at infections with low wnv titers (10 3 pfu) [95] . in our experiments, 10 5 wnv pfu were inoculated intraperitoneally. vaccinated mice had no evidence of neuronal necrosis suggesting the cd8 + t cell responses conferred protection and virus clearance. it is probable that both humoral and cellular immune responses generated against the wnv e glycoprotein prevented the virus from entering cns, potentially arresting the virus at peripheral sites. alternatively, if some virus escaped peripheral immune surveillance, it is possible that ctls cleared the virus from brain tissues before it could cause significant damage and resultant immunopathological manifestations. in summary, the vsv-e-vectored vaccine appeared to elicit robust humoral and cellular immune responses that efficiently protected mice from wnv lethal challenge. intranasal vaccination is second only to oral vaccination with regard to the relative ease of administration and patient compliance issues rendering this approach attractive for human use. recently, single-cycle vsvvectored vaccines have been shown to generate robust immune responses against a number of viral pathogens including hiv, ebola, marburg, lassa, influenza, avian influenza, hepatitis c and rsv viruses [46, 47, 49, [54] [55] [56] [57] [58] 81, 97] . based on these results, it is expected that single cycle vsv-wnv vaccines would be also efficacious. additional improvements in attenuating vsv can be made by providing more than one viral protein in trans through complementing 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and florida transmission of west nile virus from an organ donor to four transplant recipients possible dialysis-related west nile virus transmission-georgia the west nile virus and the dialysis/transplant patient west nile virus: pathogenesis and therapeutic options skin manifestations of west nile virus infection characteristics of the rash associated with west nile virus fever west nile virus neuroinvasive disease west nile virus infection: a new acute paralytic illness west nile encephalitis presenting as a stroke acute pancreatitis in west nile fever the pathology of human west nile virus infection west nile virus encephalitis with myositis and orchitis chorioretinal involvement in patients with west nile virus infection virology, pathology, and clinical manifestations of west nile virus disease the long-term outcomes of human west nile virus infection west nile virus: recent trends in diagnosis and vaccine development the anamnestic serologic response to vaccination with a canarypox virus-vectored recombinant west nile virus (wnv) vaccine in horses previously vaccinated with an inactivated wnv vaccine assessment of the efficacy of a single dose of a recombinant vaccine against west nile virus in response to natural challenge with west nile virus-infected mosquitoes in horses recombinant canarypoxvirus vaccine carrying the prm/e genes of west nile virus protects horses against a west nile virus-mosquito challenge recombinant canarypox vectored west nile virus (wnv) vaccine protects dogs and cats against a mosquito wnv challenge evaluation of the efficacy provided by a recombinant canarypox-vectored equine west nile virus vaccine against an experimental west nile virus intrathecal challenge in horses a single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against west nile virus live measles vaccine expressing the secreted form of the west nile virus envelope glycoprotein protects against west nile virus encephalitis yellow fever: an update prospects for development of a vaccine against the west nile virus efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model chimerivax-west nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy a live, attenuated recombinant west nile virus vaccine chimerivax-west nile vaccine vesicular stomatitis virus: re-inventing the bullet vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge attenuated vesicular stomatitis viruses as vaccine vectors vesicular stomatitis virus vectors expressing avian influenza h5 ha induce cross-neutralizing antibodies and long-term protection presence of bovine viral diarrhea virus (bvdv) e2 glycoprotein in vsv recombinant particles and induction of neutralizing bvdv antibodies in mice replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (rsv) antigens protect mice against rsv challenge therapeutic efficacy of vesicular stomatitis virus-based e6 vaccination in rabbits vesicular stomatitis virus-based therapeutic vaccination targeted to the e1, e2, e6, and e7 proteins of cottontail rabbit papillomavirus generation of hepatitis c virus-like particles by use of a recombinant vesicular stomatitis virus vector successful mucosal immunization of cotton rats in the presence of measles virus-specific antibodies depends on degree of attenuation of vaccine vector and virus dose properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment development of a new vaccine for the prevention of lassa fever long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type 2 gd elicit robust cd4+ th1 immune responses and are protective in mouse and guinea pig models of vaginal challenge expression of human immunodeficiency virus type 1 gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing hiv envelope a plasma membrane localization signal in the hiv-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into vsv virions specific targeting to cd4+ cells of recombinant vesicular stomatitis viruses encoding human immunodeficiency virus envelope proteins use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization an optimized vaccine vector based on recombinant vesicular stomatitis virus gives high-level, long-term protection against yersinia pestis challenge immunogenicity of attenuated vesicular stomatitis virus vectors expressing hiv type 1 env and siv gag proteins: comparison of intranasal and intramuscular vaccination routes highly effective control of an aids virus challenge in macaques by using vesicular stomatitis virus and modified vaccinia virus ankara vaccine vectors in a singleboost protocol an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants the louisiana west nile virus strain lsu-ar01 isolated from a blue jay (cyanocitta cristata) exhibits increased mouse neurovirulence in comparison to the prototypic new york-99 strain and is closely related to the connecticut-99 mosquito isolate foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus virus-specific. t cell responses in macaques acutely infected with shiv(sf162p3) west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays polyclonal activation of immature b cells by preactivated t cells: the role of il-4 and cd40 ligand cd69 is an immunoregulatory molecule induced following activation expression of interleukin (il)-2 and il-7 receptors discriminates between human regulatory and activated t cells office international des épizooties (paris) oie. vesicular stomatitis recombinant vesicular stomatitis virus as an hiv-1 vaccine vector structure of the prefusion form of the vesicular stomatitis virus glycoprotein g intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus l1 protein provides complete protection against papillomavirus-induced disease a single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1 requirement for cd40 ligand in costimulation induction, t cell activation, and experimental allergic encephalomyelitis the immune responses in cd40-deficient mice: impaired immunoglobulin class switching and germinal center formation regulation and function of class ii major histocompatibility complex, cd40, and b7 expression in macrophages and microglia: implications in neurological diseases protective capacity and epitope specificity of cd8(+) t cells responding to lethal west nile virus infection epitope discovery in west nile virus infection: identification and immune recognition of viral epitopes antigen-specific cytotoxic t lymphocytes protect against lethal west nile virus encephalitis rapid determination of hla b*07 ligands from the west nile virus ny99 genome expression of l-selectin (cd62l), cd44, and cd25 on activated bovine t cells the regulation of foxp3 expression in regulatory cd4(+)cd25(+)t cells: multiple pathways on the road cell-surface il-7 receptor expression facilitates the purification of foxp3(+) regulatory t cells cd127 expression inversely correlates with foxp3 and suppressive function of human cd4+ t reg cells cd8+ t cells mediate recovery and immunopathology in west nile virus encephalitis role of cd8+ t cells in control of west nile virus infection characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis c virus glycoproteins this work was supported by nih:ncrr 2p51rr000164 project. kgk and ai were supported by the division of biotechnology and molecular medicine (biommed), lsu school of veterinary medicine. bp was supported by the facs immunology core laboratory of the tulane national primate research center (nih:ncrr 2p51rr000164). the following reagent was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: peptide array west nile virus gene e, nr-435. we gratefully acknowledge dr. john k. rose (yale university, school of medicine) for providing us with the vsv reverse genetics systems and ms. li huang and other biommed staff for technical assistance. key: cord-296399-vvbjulm9 authors: brinkmann, constantin; hoffmann, markus; lübke, anastasia; nehlmeier, inga; krämer-kühl, annika; winkler, michael; pöhlmann, stefan title: the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells date: 2017-12-07 journal: plos one doi: 10.1371/journal.pone.0189073 sha: doc_id: 296399 cord_uid: vvbjulm9 vesicular stomatitis virus (vsv) release from infected cells is inhibited by the interferon (ifn)-inducible antiviral host cell factor tetherin (bst-2, cd317). however, several viruses encode tetherin antagonists and it is at present unknown whether residual vsv spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. here, we show that the viral glycoprotein (vsv-g) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the hiv-1 vpu protein. tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a gxxxg motif in the transmembrane domain of vsv-g. however, mutation of the gxxxg motif did not modulate tetherin sensitivity of infectious vsv. these results identify vsv-g as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread. vesicular stomatitis virus (vsv) is a negative-stranded rna virus within the rhabdoviridae family, and vsv new jersey and indiana are major vsv serotypes. vsv is transmitted from insects to ungulates (mainly cattle, horses and pigs), in which it can cause mucosal lesions [1] [2] [3] . in addition, the virus can be transmitted to humans and such infections usually induce influenza-like symptoms [3] . vsv replicates fast, is highly immunogenic and is frequently used to model infection by negative-stranded rna viruses. moreover, vsv is used as a tool for diverse scientific endeavors [4] . for instance, vsv has oncolytic properties [5] and is developed for cancer therapy [6] . moreover, vsv variants in which the open reading frame for the viral glycoprotein (vsv-g) has been replaced by that of the ebola virus (ebov) glycoprotein (gp) are currently tested as vaccines against ebov infection [7] [8] [9] . the interferon (ifn) system is an integral component of innate immunity and constitutes the first line of defense against viral infection. sensors of the ifn system, including toll-like receptors and retinoic acid inducible gene i-like receptors, can detect pathogen-associated plos one | https://doi.org/10.1371/journal.pone.0189073 december 7, 2017 1 / 19 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 molecular patterns (pamps), which triggers signals that commandeer the cells to express ifn [10, 11] . binding of ifn to uninfected cells in turn triggers further signaling events that induce the expression of ifn-stimulated genes (isg), many of which exert antiviral activity [12, 13] . vsv spread can be blocked by ifn in cell culture, although the viral matrix protein vsv-m interferes with ifn signaling [14] [15] [16] . the isg-encoded proteins that are responsible for ifninduced blockade of vsv infection are not fully known, although ifitm3 and tetherin were shown to block vsv infection in transfected cells [17, 18] . the ifn-induced antiviral host cell protein tetherin (cd317, bst-2) blocks release of diverse enveloped viruses from infected cells [19, 20] . the particular membrane topology of tetherin is key to its antiviral activity: tetherin harbors an n-terminal transmembrane domain and a c-terminal gpi-anchor which allows the protein to simultaneously insert into viral and cellular membranes, thereby forming a physical tether between virus and host cell [21] . several viruses encode tetherin antagonists which allow viral spread in tetherin-positive cells [22] . the prototypic tetherin antagonist, the hiv-1 protein vpu, and most other viral tetherin antagonists block tetherin by reducing its expression at the plasma membrane [23] [24] [25] , which is used by these viruses as platform for budding of progeny particles. in contrast, the ebov-gp, another tetherin antagonist, interferes with tetherin's antiviral activity without modulating tetherin expression or cellular localization [26] [27] [28] [29] and the mechanism underlying tetherin antagonism by ebov-gp is largely unclear. two studies reported that vsv is inhibited by tetherin. weidner and colleagues showed that directed expression of tetherin resulted in a profound decrease in vsv release from infected cells [18] . liberatore and coworkers dissected cell-cell spread of vsv from viral dissemination to distal cells via free particles and found that only the latter process was markedly inhibited by tetherin [17] . however, it is at present unknown whether vsv encodes a tetherin antagonist, which is responsible for residual viral spread in tetherin-positive cells. here, we show that vsv-g counteracts tetherin in transfected cells. however, no evidence for a contribution of tetherin-antagonism to spread of authentic vsv in tetherin-positive cells was obtained. human embryonal kidney-293t, vero (african green monkey, kidney) and hela (human, cervix carcinoma) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs, biochrome, berlin) and penicillin/streptomycin (pan-biotech, aidenach; final concentration penicillin 100 units/ml, streptomycin 0.1 µg/ml). bhk-21 cells (baby hamster kidney) were cultivated in dmem supplemented with 5% fbs (biochrome) and penicillin/streptomycin. cells were cultured at 37˚c in humidified atmosphere containing 5% co 2 . for seeding and subcultivation, cells were washed with phosphatebuffered saline (pbs) and detached by incubation in a trypsin/edta solution (pan-biotech, aidenach; hela, vero and bhk-21 cells) or by directly resuspending them in dmem (293t). cell numbers were determined under a light microscope using a neubauer chamber. transfection of vero, bhk-21 and hela cells was performed using lipofectamine2000 (thermofisher scientific, dreieich) according to manufacturer's protocol, while 293t cells were transfected using the calcium phosphate method. selection and maintenance of vero cells stably transfected with the retroviral pqcxip vector was achieved using puromycin (cayman chemical, ann arbor). 293t cells were obtained from dsmz (acc-635, leibniz institute dsmz-german collection of microorganisms and cell cultures). vero and bhk-21 cells were obtained from collaborators. hela cells were obtained through the nih aids reagent program, division of aids, niaid, nih and their identity was confirmed by str dna typing employing a published protocol [30, 31] . plasmids encoding vpu, ebov-gp and hiv-1 p55-gag were previously described [27, 32] . expression plasmids for the coding sequences of vsv nucleoprotein (vsv-n), phosphoprotein (vsv-p), matrix protein (vsv-m), glycoprotein (vsv-g) and rna-dependent rnapolymerase (vsv-l) were generated by amplifying the respective open reading frame (orf) from a plasmid-encoded vsv anti-genome (indiana strain, kindly provided by g. zimmer) and inserting them via standard cloning procedures into plasmids pcaggs (vsv-n, vsv-p, vsv-m (mutant ncp, harboring four amino acid substitutions associated with reduced cytotoxicity [33] ) and vsv-l) or pcg1 (vsv-g). this was achieved by overlap-extension pcr technique using primers that introduce the desired nucleotide exchanges. generation of vsv-g mutants a133r and lxxxl was achieved by the same strategy using the expression plasmid for wt vsv-g as a template. the expression plasmid for human tetherin was generated by amplifying the orf from a previously described plasmid [34] and inserting it into plasmid pcaggs using kpni and xhoi restriction sites. similarly, porcine tetherin was pcr amplified and cloned via ecori and nhei restriction sites. for detection of human tetherin expression, the sequence for a c-myc antigenic tag was added to the 5' end of human tetherin using pcr. in order to generate transgenic cells stably expressing human tetherin, the genetic information for human tetherin was cloned into the retroviral vector pqcxip-mcs, a modified form of pqcxip (clontech, palo alto), in which the multiple cloning site was modified to contain sites for noti-bamhi-agei-hpai-mlui-xhoi-nrui-ecori. to generate recombinant vsv that expresses egfp (enhanced green fluorescent protein), we constructed a plasmidencoded vsv anti-genome (genbank: j02428.1) with an additional transcription unit for egfp between vsv-g and vsv-l orfs. first, unique mlui and nhei restriction sites were introduced upstream and downstream of the orf for vsv-g, respectively. next, a cassette consisting of the coding sequences of vsv-g and egfp, separated by a minimal intergenic region [35] was cloned and inserted into the parental vsv genome making use of mlui and nhei restriction sites, thereby replacing the genetic information of vsv-g and thus generating vsv ã (the asterisk stands for egfp). for convenient exchange of vsv-g or egfp orfs by other transcription units, unique asci and noti restriction sites were added upstream and downstream of the vsv-g and egfp orfs, respectively. in order to generate vsv ã mutant vsv-g (lxxxl), the respective orf was amplified from the corresponding vsv-g (lxxxl) expression plasmids with primers adding 5' mlui and 3' asci restriction sites and inserted into vsv ã , thereby replacing the parental orf coding for wt vsv-g. the integrity of all pcramplified sequences was confirmed by automated sequencing. release of virus-like particles (vlps) and its inhibition by tetherin has been examined as described [27, 32] . in brief, 293t cells were seeded in 6-well plates and cotransfected with plasmids encoding hiv-1 p55-gag, tetherin and a potential tetherin antagonist or empty plasmid, using the calcium phosphate method. at 16 h post transfection, the transfection medium was replaced by fresh culture medium. for blockade of vsv-g-dependent tetherin antagonism, anti-vsv-g hybridoma supernatant (i1, concentrated mouse hybridoma supernatant from crl-2700, atcc) was added to the culture medium at a final dilution of 1:1,000. at 48 h post transfection the supernatants were collected and cells were lysed in 200 μl of 2x sds-containing lysis buffer (30 mm tris [ph 6.8], 10% glycerol, 2% sds, 5% β-mercaptoethanol, 0.1% bromophenol blue, 1mm edta). the lysates were incubated at 95˚c for 30 min. the supernatants were cleared by centrifugation and vlps were pelleted from cleared supernatants by centrifugation through a 20% sucrose cushion. the concentrated vlps were lysed in 50 μl 2 x sds loading buffer and incubated at 95˚c for 30 min. subsequently, cell lysates and supernatants were analyzed for the presence of gag employing western blot. for immunoblotting, the proteins were separated via sds-polyacrylamid (paa) gel electrophoresis using a 12.5% paa gel and transferred onto a nitrocellulose membrane (ge healthcare life sciences, solingen, 0.2 μm). the membranes were blocked in 5% milk powder in pbs supplemented with 0.1% tween 20 and gag-protein was detected using 1:100 diluted supernatants of hybridoma cells secreting a mouse anti-gag antibody (183-h12-5c). expression of vsv-g wt and mutants was detected using the aforementioned anti-vsv-g hybridoma supernatant at a dilution of 1:100 while vsv-m was detected using mouse monoclonal antibody 23h12 (kerafast, boston). tetherin expression was detected employing anti c-myc-hybridoma supernatants (c-mycl-9e10 (ecacc 85102202)). expression of vsv proteins, vpu and ebov-gp was detected using previously described rabbit sera [36] [37] [38] . expression of ß-actin was detected using mouse anti-ß-actin antibody (sigma-aldrich, munich) at a dilution of 1:1,000. bound antibodies were detected using hrp-coupled anti-mouse and anti-rabbit secondary antibodies (dianova, hamburg) at a final concentration of 0.1 μg/ml. binding of secondary antibodies was detected using a self-made ecl solution (0.1 m tris-hcl ph 8.6, 250 μg/ml luminol (sigma-aldrich, münchen), 1 mg/ml para-hydroxycomaric acid (sigma-aldrich, münchen); 0.3% h 2 o 2 ) and signals were visualized using the chemocam imaging system and the chemostar-professional software (intas). for quantification of the signal intensity, the program imagej (fiji distribution) [39] was used. gag signals detected in the supernatants were normalized against the respective signals obtained in cell lysates. for the rescue of vsv ã harboring wt or mutant vsv-g, we employed a strategy developed by others [40] with slight modifications. first, bhk-21 cells were grown in 12-well dishes until they reached~70% confluency. at this point, the cells were infected with recombinant modified vaccinia virus ankara expressing t7 polymerase ( [41] , vmva-t7, kindly provided by g. sutter) at a multiplicity of infection (moi) of 3. at 1 h post infection, the inoculum was removed and the cells were transfected with expression plasmids for vsv-n, -p and -l (see above) as well as the respective, plasmid-encoded vsv ã anti-genome (the genome is preceded by a t7 promotor sequence and followed by a hepatitis delta virus ribozyme and a t7 terminator sequence for cytoplasmic production of negative-sensed viral genomes that are template for transcription and genome replication) using lipofectamine2000 (thermofisher scientific, dreiech) as transfection reagent. the following dna concentrations were used (per well): 0.4 μg vsv-n, 0.35 μg vsv-p, 0.25 μg vsv-l and 1.0 μg plasmid-encoded vsv ã or vsv ã (lxxxl) genome. transfection was carried out in optimem medium (thermofisher scientific, dreiech) for 6 h, before the transfection medium was replaced by standard culture medium. at 12 h post transfection, the culture medium was replaced by medium containing 100 μg/ml rifampicin and 40 μg/ml cytosine β-d-arabinofuranoside (both from sigma-aldrich, münchen) to limit rmva-t7 replication. after an additional 24 h, the culture medium was collected, clarified from cellular debris by centrifugation (4,700 rpm, 10 min, 4˚c) and twice filtered through a membrane filter with a pore size of 0.2 μm to eliminate rmva-t7 from the preparation. next, fresh bhk-21 cells grown in t-75 flasks were inoculated with a 1:100 dilution of the passage 0 (p0) to amplify vsv ã for virus stock production (p1). quantification of titers from vsv ã stocks and cell culture supernatant was carried out on confluently grown bhk-21 cells seeded in 96-well plates. after removal of the cell culture supernatant, cells were inoculated with 10-fold serial dilutions of virus (diluted in serum-free medium). at 1 h post infection, the inoculum was removed and cells were overlaid with culture medium containing 2% methylcellulose (sigma-aldrich, münchen). after an incubation of 16-18 h, egfp-positive cells were counted under the fluorescence microscope to determine viral titers (displayed as focus forming units per ml, ffu/ml). in addition, vsv ã titers were also determined for hela cells as this cell line is less susceptible to vsv infection (~100x) and therefore titers determined on bhk-21 cells are not useful to calculate the optimal infectious dose for hela cells. to verify the authenticity of wt and mutant vsv-g in vsv ã , viral rna was isolated from p1 stocks and reverse-transcribed into cdna using the superscript iii first-strand synthesis system (thermofisher scientific, dreiech) according to the manufacturer's protocol (for random hexamers). next, a~1,800 bp fragment was amplified with primers binding in the intergenic region upstream of vsv-g (forward) and the 5' end of the egfp orf (reverse) using phusion polymerase (thermofisher scientific, dreiech), separated by agarose gel electrophoresis and extracted from the gel by commercial kits (macherey & nagel, düren), before being subjected to automated sequence analysis (seqlab, göttingen). to assess the effect of directed tetherin expression on the spread of vsv ã , 293t cells were transfected with increasing amounts of expression plasmid encoding for human tetherin. to avoid unspecific effects due to differences in the total dna amounts of the tetherin vector, empty expression plasmid was used for equilibration. cells transfected only with empty expression vector served as controls. at 16 h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for 24 h before they were infected. additionally, vero cells stably expressing human tetherin (vero-tetherin) or stably containing empty vector were used. in order to analyze the impact of sirna-mediated knockdown of endogenous tetherin expression on vsv spread, hela cells were transfected with sirna specific to human tetherin or scrambled sirna (control) (both santa cruz, dallas; 10 pm final concentration) using lipofectamine2000 and optimem medium (both from ther-mofisher scientific, dreieich). at 6 h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for 24 h before they were infected. for infection, vsv ã stocks were diluted with serum-free medium to obtain the desired moi and inoculated onto target cells for 1 h. afterwards, the inoculum was removed and cells were washed with pbs before receiving fresh culture medium. next, cells were further incubated and supernatants were collected for quantification of vsv ã titers at different time points. to analyze vsv-g-driven host cell entry, vsv vectors were pseudotyped with either vsv-g wt or mutants a133r and lxxxl. for this, 293t cells were transfected with the respective expression plasmids or empty plasmid as negative control. at 24 h post transfection, cells were inoculated with vsv-g trans-complemented vsv ã δg that lacks the genetic information for vsv-g but codes for egfp and firefly luciferase from two independent transcription units [42] (kindly provided by g. zimmer) at an moi of 3. at 1 h post infection, the inoculum was removed and the cells were washed with pbs. next, medium containing a neutralizing antibody directed against vsv-g (i1, produced from crl-2700 hybridoma cells, atcc) was added to the cells and left for 1 h in order to neutralize residual input virus that had not entered the cells so far. subsequently, the cells were again washed and further incubated with fresh culture medium for 16-18 h. then, the supernatant was collected, clarified from cellular debris by centrifugation (4,700 rpm, 10 min, 4˚c) and used for transduction experiments. for this, 293t cells were inoculated with identical volumes of the respective vsv pseudotypes and firefly luciferase activity in cell lysates was quantified at 20 h post inoculation using a plate luminometer (hidex) and commercial substrates (pjk and promega) as described elsewhere [43] . for analysis of the surface expression of tetherin, 293t cells were cotransfected with tetherin plasmid and plasmid encoding viral antagonist or empty plasmid as negative control. at 48 h post transfection, the cells were washed and harvested in pbs. expression of tetherin at the cell surface was detected by employing a tetherin-specific mouse monoclonal antibody (biolegend) at a dilution of 1:50 and an alexa 647-conjugated anti-mouse secondary antibody at a dilution of 1:100 (thermofisher scientific, dreieich). subsequently, cells were fixed with 2% paraformaldehyde and staining was analyzed employing a lsr ii flow cytometer (bd biosciences, heidelberg) and the facs diva software (bd biosiences, heidelberg). the data were further analyzed using the fcs express 4 flow research software (de novo software). to ensure that directed tetherin expression did not lead to unwanted cytotoxic side-effects at the concentrations used, we analyzed cell viability employing the celltitre-glo kit (promega, mannheim). for this, 293t cells grown in 12-well plates were transfected with increasing amounts of tetherin expression plasmid or empty vector, which was also used to equilibrate total dna amounts. at 16 h post transfection, the transfection medium was replaced by fresh culture medium and the cells were further incubated for 24 h. next, the cell culture supernatants were removed and 200 μl of the celltitre-glo reagent were added. after an incubation period of 10 min at room temperature, 100 μl of the lysates were transferred into a white, opaque-walled 96-well plate, before luminescence was recorded using a plate luminometer (plate chameleon v, hidex, turku). all samples were analyzed in triplicates. for normalization, viability of cells transfected only with empty expression vector was set as 100% and relative viability of tetherin-transfected cells was calculated. if not explicitly stated in the figure legends, unpaired and paired student t-tests were performed to analyze data pairs originating from individual experiments or mean data of multiple experiments, respectively. one-way anova with bonferroni post-test analysis was carried out for combined comparison of multiple groups ( ã , p 0.05; ãã , p 0.005; ããã , p 0.001). in order to analyze tetherin antagonism by vsv proteins, we employed a previously reported virus-like particle (vlp) release assay, which measures release of hiv-gag-based vlps from transfected 293t cells and its blockade by tetherin [27, 32] . release of vlps was markedly diminished upon coexpression of tetherin and tetherin's antiviral activity was blocked by the well-established tetherin antagonists vpu and ebov-gp (fig 1a and 1b) , as expected. in addition, vsv-g but not vsv-l, m (mutant m(ncp), [33] ) and p interfered with tetherin's antiviral activity, although less efficiently than vpu and ebov-gp (fig 1a and 1b) . finally, none of the viral proteins tested modulated vlp release from tetherin-negative control cells, indicating that the effects observed in tetherin-positive cells were specific. thus, vsv-g can antagonize tetherin in transfected cells, although with reduced efficiency as compared to ebov-gp and vpu. the studies described above were conducted with human tetherin. since vsv can infect livestock, we next analyzed if vsv-g also antagonizes pig-derived tetherin. we found that vpu failed to antagonize porcine tetherin (fig 1c) , in keeping with the well-established notion that the anti-tetherin activity of vpu is highly species specific [44] [45] [46] . in contrast, both ebov-gp and vsv-g rescued vlp release from blockade by porcine tetherin (fig 1c) , indicating that vsv-g might be able to promote viral spread in infected pigs by antagonizing tetherin. evidence that tetherin antagonism by vsv-g can be blocked by a gprotein specific antibody and is not due to vsv-g overexpression we next sought to investigate whether tetherin counteraction by vsv-g can be inhibited by antibodies. this question was triggered by our previous observation that antibodies directed against ebov-gp can interfere with gp-mediated tetherin counteraction [32] . to this end, we added supernatant of hybridoma crl-2700 (which secretes the vsv neutralizing antibody i1) to cells expressing vsv-g and releasing vlps in the presence and absence of tetherin. the hybridoma supernatant did not modulate vlp release from tetherin-negative control cells coexpressing vsv-g but abrogated vlp-release from cells coexpressing tetherin and vsv-g (fig 1d and 1e) . we cannot exclude that the antibody triggered vsv-g internalization and a control antibody remains to be tested. however, the results available at present suggest that antibodies directed against vsv-g might not only block viral entry into target cells but may also interfere with tetherin antagonism. moreover, our findings indicate that the ectodomain of vsv-g plays an important role in tetherin counteraction. we next investigated whether the vsv-g expression levels attained in transfected cells exceeded those found in infected cells, which would suggest that tetherin counteraction by vsv-g could have been due to overexpression. for this, vsv-g expression levels in cells transfected to express vsv-g and cells infected with a recombinant vsv encoding gfp (vsv ã ) were compared. cells were harvested at the same time points at which vlp release and (figs 1a-1e and 5e and 5f) and vsv release (fig 3a-3c and fig 6a-6d ) from tetherin-positive cells were examined within functional assays. the immunoblot revealed that less g-protein was expressed in transfected as compared to infected cells (fig 1f) , indicating that tetherin antagonism was not due to overexpression. in this context, it should be noted that the lack of correlation between infectious dose and g-protein expression levels resulted from the fast replication kinetics of vsv, which led to 100% infected cells at the time of analysis. moreover, the reduced arrow heads indicate bands exhibiting the molecular weight expected for unglycosylated (white), partially (grey) and fully (black) n-glycosylated tetherin. (c) quantification of four experiments performed as described for panel (b). the intensities measured for all tetherin signals with a molecular weight between 17 and 30 kda were added to yield total tetherin expression. tetherin expression in the absence of antagonist (mock) was set to 100%. error bars indicate sem. one-way anova with bonferroni post-test analyses were performed for panels a and c to test for statistically significant differences between samples with and without (mock) antagonist (*, p 0.05). signals for actin in vsv-versus mock-infected cells can be attributed to the well-established cytotoxic effects associated with vsv infection [33, 47, 48] . collectively, the results discussed tetherin antagonism by viral proteins is usually due to removal of tetherin from the site of viral budding, the plasma membrane [20, 22] . in order to investigate whether vsv-g also interferes with tetherin expression at the plasma membrane, we performed flow cytometry with cells transfected to coexpress tetherin and viral antagonists. coexpression of vpu but not ebov-gp reduced tetherin surface levels (fig 2a) , as expected from previous reports [27, 28] . in contrast, coexpression of vsv-g did not modulate surface levels of tetherin (fig 2a) . in agreement with these findings, neither vsv-g nor ebov-gp coexpression reduced tetherin levels in cell lysates while a marked decrease in tetherin levels was observed upon coexpression of vpu (fig 2b and 2c) . thus, vsv-g, like ebov-gp, employs a mechanism different from that of vpu for tetherin counteraction. upon coexpression of vsv-g and ebov-gp, a band with the size expected for unglycosylated tetherin accumulated (fig 2b) , which is known to exert reduced antiviral activity as compared to the fully glycosylated form [21] . therefore, it is conceivable that vsv-g and ebov-gp inhibit tetherin by interfering with its n-glycosylation, similarly as reported for sars-coronavirus orf7a protein [49] . weidner and colleagues reported that vsv spread can be efficiently blocked by directed expression of tetherin [18] , and a subsequent study demonstrated that tetherin mainly inhibits cell-free but not cell-associated vsv spread [17] . we sought to confirm these findings and to establish a cellular system which allows investigating the potential contribution of vsv-gmediated tetherin counteraction to viral spread. for this, we transfected 293t cells with rising amounts of tetherin plasmid, infected the cells with vsv and quantified the number of infectious units present in culture supernatants at 8 h post infection. we observed a modest but dose-dependent reduction of viral titers upon transfection of increasing amounts of tetherinplasmid (fig 3a) in the absence of appreciable cytotoxic effects (s1 fig). moreover, sirnamediated knock-down of endogenous tetherin expression in hela cells reduced the amount of cell surface associated tetherin (fig 3b) and increased viral titers roughly 10-fold as compared to cells transfected with scrambled sirna or mock transfected cells (fig 3c) . collectively, these findings confirm that vsv is inhibited by tetherin and indicate that knock-down of tetherin expression in hela cells affords the opportunity to examine whether vsv-g-mediated tetherin antagonism is operative in the context of infected cells. program is presented. expression of vsv-g wt was set to 100%. (c) incorporation of vsv-g wt and mutant lxxxl into vsv pseudotypes was investigated by western blot analysis using an anti-vsv-g antibody (concentrated supernatants from hybridoma cl-2700). to ensure that similar amounts of pseudotypes were analyzed, levels of particle-associated m proteins were determined using an anti-vsv-m antibody. pseudotypes harboring no glycoprotein (mock) served as negative control. the results of a single immunoblot are shown from which irrelevant lanes were cut out. similar results were obtained in two separate experiments. (d) 293t cells were transduced with equal volumes of the vsv pseudotypes described in panel (c). at 24 h post transduction, luciferase activity in cell lysates was measured. transduction driven by vsv-g wt was set as 100%. the average of three independent experiments is shown. error bars indicate sem. (e) 293t cells were cotransfected with plasmids encoding gag, the indicated glycoproteins and tetherin. expression of gag in supernatants and cell lysates was determined by western blot. detection of β-actin expression served as loading control. (f) the average of three independent experiments conducted as described for panel (e) and quantified via the imagej program is presented. error bars indicate standard error of the mean (sem). release of gag from cells coexpressing the highest amount of vsv-g and tetherin was set to 100%. for all graphs (b, d, f), paired two-tailed students' t-tests were performed to assess whether differences between vsv-g wt and lxxxl mutant were of statistical significance. https://doi.org/10.1371/journal.pone.0189073.g005 a gxxxg motif in the transmembrane domain of vsv-g contributes to tetherin antagonism in transfected cells but is dispensable for vsv spread in tetherin-positive cells having established that vsv-g can antagonize tetherin upon directed expression and that vsv spread is reduced but not abrogated by tetherin, we finally sought to determine whether vsv-g-mediated tetherin antagonism contributes to viral spread. for this endeavor, we needed to identify a mutation in vsv-g that selectively interferes with tetherin antagonism. to this end, we tested a vsv-g mutant in which a gxxxg motif in the transmembrane domain was changed to lxxxl (fig 4) . this mutation was chosen, since our unpublished results indicate that a gxxxa motif in the transmembrane domain of ebov-gp contributes to tetherin antagonism. in addition, we analyzed vsv-g mutant a133r. this mutation is known to interfere with viral entry by rendering vsv-g defective for membrane fusion [50] . both mutation a133r and mutation of the gxxxg motif (mutant lxxxl) were compatible with robust g-protein expression (fig 5a and 5b ) and particle incorporation (fig 5c) . moreover, mutation of the gxxxg motif (mutant lxxxl) did not interfere with host cell entry of vsv-g pseudotypes (fig 5d) . in contrast, mutation a133r markedly reduced entry efficiency (fig 5d) , as expected [50] . finally, mutant a133r was able to counteract tetherin while mutant lxxxl failed to efficiently antagonize tetherin (fig 5e and 5f ). thus, mutation of the gxxxg motif afforded an opportunity to investigate whether g-protein-mediated tetherin antagonism contributes to viral spread. for this, the lxxxl mutation was introduced into the vsv genome, employing a reverse genetics system, and infectious vsv was rescued. infection of sirna-transfected hela cells with vsv wt and mutant lxxxl at equal moi revealed that knock-down of tetherin expression increased vsv wt release, as expected. however, a comparable rescue of viral release was observed for the lxxxl mutant (fig 6a and 6b) . moreover, spread of vsv wt and mutant lxxxl was comparably reduced by directed expression of tetherin in vero cells (fig 6c and 6d) , suggesting that tetherin antagonism by vsv-g might not appreciably contribute to viral spread in tetherin-positive cells. tetherin and other effector proteins of the ifn system are responsible for the establishment of an antiviral state in ifn exposed cells [12, 13] . many viruses evolved countermeasures against these antiviral effectors by either multi-functionalizing their structural proteins or by acquiring non-structural proteins with antagonistic activity. whether vsv, which is sensitive to blockade by tetherin [17, 18] , encodes an antagonist that allows residual viral spread in tetherin-positive cells is unknown. here, we show that the surface protein of vsv, vsv-g, can antagonize tetherin, at least upon directed expression. tetherin counteraction by vsv-g did not involve modulation of tetherin expression and was partially dependent on a gxxxg motif in the vsv-g transmembrane domain. however, mutation of the gxxxg motif in the context of infectious vsv did not affect viral spread in tetherin-positive cells. thus, vsv-g is a novel tetherin antagonist but the potential contribution of its antagonistic activity to viral spread in tetherin-positive cells requires further investigation. previous studies reported that directed expression of tetherin in nih 3t3 cells and 293 cells markedly diminished viral release [17, 18] . however, tetherin expression failed to reduce viral release to background levels in both studies, although tetherin was expressed at high levels, which leaves the possibility that vsv encodes a modestly active tetherin antagonist. in keeping with such a scenario, we found that vsv-g antagonizes human and pig tetherin but does so less efficiently than vpu and ebov-gp. tetherin antagonism could only be demonstrated in the context of directed vsv-g expression in the present study. nevertheless, tetherin counteraction was not due to g-protein overexpression, indicating that vsv-g could block tetherin in infected cells, as discussed below. several viral tetherin antagonists inhibit tetherin by interfering with its expression or appropriate cellular localization. for instance, vpu can interfere with the anterograde transport of tetherin [51, 52] and directs tetherin towards lysosomal degradation [23] [24] [25] , which ultimately results in reduced tetherin levels at the cell surface. in contrast, vsv-g did not modulate total tetherin expression or tetherin levels at the cell surface. these findings are similar to those previously reported for ebov-gp [27, 28] , which antagonizes tetherin by a so far unknown mechanism. moreover, vsv-g, like ebov-gp, was active against diverse tetherins, as demonstrated by the counteraction of porcine tetherin, which shares only 48% sequence identity with human tetherin. finally, our finding that antibody i1 directed against the vsv-g ectodomain interferes with tetherin antagonism recapitulates findings previously made for ebov-gp [32] and suggests that both vsv-g and ebov-gp depend on their ectodomains for tetherin counteraction. how vsv-g (and ebov-gp) counteracts tetherin remains unknown and at present a role of direct interactions (potentially disrupted by anti-ectodomain antibodies) cannot be discounted. our finding that coexpression of vsv-g resulted in increased generation of unglycosylated tetherin, which is known to display little antiviral activity [21] , suggests that vsv-g might block tetherin, at least in part, by interfering with its posttranslational modification. a similar inhibitory strategy has previously been reported for the sars-coronavirus orf7a protein [49] but the underlying mechanism is incompletely understood. in order to get initial insights into the contribution of vsv-g-mediated tetherin antagonism to vsv release from tetherin-positive cells, we examined previously characterized mutations in the ectodomain (a133r) and the transmembrane domain (g473l and g477l, mutant lxxxl) of vsv-g for their effect on tetherin antagonism. a133r markedly reduced vsv-gdriven entry, as expected [50] , but did not affect tetherin antagonism. in contrast, mutation of the gxxxg motif in the transmembrane domain had no impact on entry but reduced tetherin antagonism by roughly 60%, as discussed below. the finding that the gxxxg motif was dispensable for vsv-g-driven virus-cell fusion was unexpected, since the same motif has been reported to be required for cell-cell fusion induced by exposure of vsv-g expressing cells to low ph [53] . in this context, it is important to state that the integrity of the lxxxl mutations after amplification of the rescued viruses in bhk-21 cells (passage 1 stocks) has been confirmed by sequencing. therefore, the gxxxg motif seems to be important for vsv-g-driven cell-cell but not virus-cell fusion and the underlying reasons remain to be determined. the finding that the gxxxg motif is required for tetherin antagonism is in keeping with our unpublished observation that a gxxxa motif in the ebov-gp transmembrane domain, which has been reported to be important for ebov-gp-mediated cellular detachment [54] , is required for full tetherin antagonism by ebov-gp. although mutation of the gxxxg motif in vsv-g reduced tetherin antagonism in transfected cells, it had no impact on viral spread in hela cells, which express endogenous tetherin [19] , and vero cells, which were engineered to express tetherin. these observations could indicate that vsv-g-mediated tetherin antagonism is not operative in vsv infected cells. in fact, one could speculate that in infected cells the interaction between vsv-g and other viral proteins, which is required for assembly of progeny particles [55] , compromises the ability of vsv-g to counteract tetherin. alternatively, it is conceivable that the modest reduction of tetherin antagonism observed in transfected cells upon mutation of the gxxxg motif precluded detection of anti-tetherin effects in infected cells. the identification of a mutation in vsv-g that selectively and efficiently reduces tetherin antagonism is required to address this question. collectively, our results and published data indicate that vsv-g, ebov-gp [26] and possibly other viral glycoproteins can interfere with tetherin's antiviral activity via a mechanism that is relatively independent of the tetherin sequence. glycoprotein-mediated interference with tetherin's n-glycosylation status might contribute to this effect. moreover, our results indicate that findings made for tetherin-antagonism in transfected cells might not always adequately reflect the situation in infected cells. finally, our findings suggest the possibility that vsv might acquire robust tetherin antagonism upon infection of humans. supporting information s1 fig. directed tetherin expression is not associated with major cytotoxic effects. 293t cells were transfected with increasing amounts of expression vector for human tetherin. empty vector was used for equilibration of total dna amounts. at 48 h post transfection, intracellular atp levels were quantified. the average of three independent experiments performed with triplicate samples is shown, error bars indicate standard error of the mean (sem). a paired two-tailed student's t-test was used to examine whether differences in cell viability between cells transfected with empty vector (0 μg tetherin plasmid) and tetherin-expressing cells were of statistical significance (ns, not significant; ã , p 0.05). (pdf) vesicular stomatitis transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice emergence and re-emergence of vesicular stomatitis in the united states vesicular stomatitis virus: re-inventing the bullet oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, ras, or myc function and involves the induction of apoptosis oncolytic vesicular stomatitis virus as a viro-immunotherapy: defeating cancer with a "hammer" and "anvil assessing the safety and immunogenicity of recombinant vesicular stomatitis virus ebola vaccine in healthy adults: a randomized clinical trial six-month safety data of recombinant vesicular stomatitis virus-zaire ebola virus envelope glycoprotein vaccine in a phase 3 double-blind, placebo-controlled randomized study in healthy adults safety and immunogenicity of the rvsvg-zebov-gp ebola virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, placebo-controlled, dose-response study prrs are watching you: localization of innate sensing and signaling regulators pattern recognition receptors and the innate immune response to viral infection interferon-stimulated genes: a complex web of host defenses a diverse range of gene products are effectors of the type i interferon antiviral response the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter factors affecting the sensitivity of different viruses to interferon rhabdovirus evasion of the interferon system tetherin inhibits cell-free virus dissemination and retards murine leukemia virus pathogenesis interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the antiviral activities of tetherin tetherin inhibits hiv-1 release by directly tethering virions to cells counteraction of the multifunctional restriction factor tetherin differential effects of human immunodeficiency virus type 1 vpu on the stability of bst-2/tetherin vpu directs the degradation of the human immunodeficiency virus restriction factor bst-2/tetherin via a {beta}trcp-dependent mechanism vpu antagonizes bst-2-mediated restriction of hiv-1 release via beta-trcp and endo-lysosomal trafficking tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein the ebola virus glycoprotein and hiv-1 vpu employ different strategies to counteract the antiviral factor tetherin ebola virus glycoprotein counteracts bst-2/tetherin restriction in a sequence-independent manner that does not require tetherin surface removal anti-tetherin activities of hiv-1 vpu and ebola virus glycoprotein do not involve removal of tetherin from lipid rafts recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues short tandem repeat typing technologies used in human identity testing the tetherin antagonism of the ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by gp1-specific antibodies fusionactive glycoprotein g mediates the cytotoxicity of vesicular stomatitis virus m mutants lacking host shutoff activity tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv-1 strains the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes human immunodeficiency virus type 1 vpu protein is an oligomeric type i integral membrane protein modulation of virion incorporation of ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization fiji: an opensource platform for biological-image analysis recombinant vesicular stomatitis viruses from dna non-replicating vaccinia vector efficiently expresses bacteriophage t7 rna polymerase a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon the glycoproteins of all filovirus species use the same host factors for entry into bat and human cells but entry efficiency is species dependent hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor species-specific activity of siv nef and hiv-1 vpu in overcoming restriction by tetherin/bst2 species-specific activity of hiv-1 vpu and positive selection of tetherin transmembrane domain variants the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 severe acute respiratory syndrome coronavirus orf7a inhibits bone marrow stromal antigen 2 virion tethering through a novel mechanism of glycosylation interference molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein g, a class iii viral fusion protein hiv-1 vpu antagonizes bst-2 by interfering mainly with the trafficking of newly synthesized bst-2 to the cell surface hiv-1 vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor cd317/tetherin to overcome the virion release restriction the transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus g protein inhibition of ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein the following reagents were obtained through the nih aids reagent program, division of aids, niaid, nih: anti-hiv-1 nl4-3 vpu polyclonal antiserum from dr. klaus strebel, hela cells from dr. richard axel. polyclonal antiserum against vsv was kindly provided by dr. georg herrler. key: cord-020714-h1fevqcw authors: compans, richard w.; kemp, maurice c. title: membrane glycoproteins of enveloped viruses date: 2008-05-30 journal: curr top membr transp doi: 10.1016/s0070-2161(08)60750-9 sha: doc_id: 20714 cord_uid: h1fevqcw this chapter focuses on the recent information of the glycoprotein components of enveloped viruses and points out specific findings on viral envelopes. although enveloped viruses of different major groups vary in size and shape, as well as in the molecular weight of their structural polypeptides, there are general similarities in the types of polypeptide components present in virions. the types of structural components found in viral membranes are summarized briefly in the chapter. all the enveloped viruses studied to date possess one or more glycoprotein species and lipid as a major structural component. the presence of carbohydrate covalently linked to proteins is demonstrated by the incorporation of a radioactive precursor, such as glucosamine or fucose, into viral polypeptides, which is resolved by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. enveloped viruses share many common features in the organization of their structural components, as indicated by several approaches, including electron microscopy, surface-labeling, and proteolytic digestion experiments, and the isolation of subviral components. the chapter summarizes the detailed structure of the glycoproteins of four virus groups: (1) influenza virus glycoproteins, (2) rhabdovirus g protein, (3) togavirus glycoprotein, and (4) paramyxovirus glycoproteins the information obtained includes the size and shape of viral glycoproteins, the number of polypeptide chains in the complete glycoprotein structure, and compositional data on the polypeptide and oligosaccharide portions of the molecules. search. the most important advantage is structural simplicity, in that lipid-containing viruses possess a small number of virus-coded polypeptides and glycoproteins as membrane components. sufficient quantities of many enveloped viruses can be purified for detailed biochemical analysis, including primary structure studies of viral membrane proteins like those now underway. purification procedures are rapid and simple, and a fairly homogeneous population of particles can be obtained free of contaminating cellular membranes. for the simpler lipid-containing viruses, the possibility therefore exists for complete structural determination of a biological membrane, and such information should contribute greatly to an understanding of the molecular details of membrane structure and interactions among membrane components in general. other advantages of enveloped viruses in studies of membrane structure and biogenesis include the ease of biosynthetic labeling of viruses grown in cell culture with specific radioactive precursors and the availability of mutants in defined gene products, some of which are proving to be useful in the analysis of viral membrane assembly. since envelope components are integral parts of cellular membranes during the assembly of virus particles (compans et al., 1966 ; compans and choppin, 1971) , studies of the synthesis and mechanism of insertion of these components into membranes should provide useful information pertaining to the biogenesis of cellular membranes. many viral systems offer the additional advantage that host cell biosynthesis is inhibited during infection, so that it is possible to analyze the synthesis of membrane components in a cell in which only a small number of virus-coded membrane components are being produced and to follow the intracellular migration of specific membrane glycoproteins by radiolabeling procedures. finally, the ability to prepare viruses with specific modifications in either lipid or protein composition arises from the fact that viral envelope lipids are derived from the plasma membrane of the host cell, whereas viral proteins are entirely virus-coded. thus by growing different virus types in the same cell it is possible to prepare a membrane in which the protein composition can be varied while the lipid composition remains constant. similarly, by growing the same virus in a series of different cells, viral membranes are obtained which possess the same set of proteins but vary in lipid composition. these approaches have been useful in studies of lipid-protein interactions in viral membranes (landsberger et al., 1973) . several recent reviews are available concerning both the experimental and theoretical aspects of viral envelope structure and assem-bly (lenard and compans, 1974 ; blough and tiffany, 1975 ; wagner, 1975 ; choppin and compans, 1975 ; compans and choppin, 1975 ; klenk, 1974) . in this chapter, we emphasize recent information on the glycoprotein components of enveloped viruses and endeavor to point out specific findings on viral envelopes which we believe to be of broad significance. in table i are listed the 10 established major groups, or families, of lipid-containing viruses of vertebrates, and examples of the best studied members of certain groups. for several families, only limited information is available on viral envelope structure, whereas other groups have been the object of intensive study. we concentrate our discussion on the glycoproteins of the most intensively studied virus groups, which are the first four groups of rna viruses listed in table i : myxoviruses, paramyxoviruses, rhabdoviruses, and togaviruses. we also discuss selected findings for other virus groups where these have revealed unusual or important aspects of viral membrane structure or assembly. although enveloped viruses of different major groups vary in size and shape, as well as in the mws of their structural polypeptides, there are general similarities in the types of polypeptide components present in virions. the types of structural components found in viral membranes are summarized briefly in the following discussion, and further detailed information is available in the recent reviews cited above. all the enveloped viruses studied to date possess one or more glycoprotein species. the presence of carbohydrate covalently linked to proteins has usually been demonstrated by the incorporation of a radioactive precursor such as glucosamine or fucose into viral polypeptides, which is resolved by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. the specificity of labeling by these precursors is shown by the lack of their incorporation into carbohydrate-free polypeptides of the virion (straws et al., 1970) . the number of distinct glycoprotein species present in the virion varies for various virus groups. as shown in table 11 , there is also marked variation in the glycoprotein content of virions when expressed as a percent of (gard et al., 1977) , whereas the data given are for pichinde virions which contain two glycoproteins. the values represent our calculations from the autoradiograph scan data presented in the reference. the values given represent our calculations from the data presented in the reference. total viral protein. as discussed in section v, these data suggest that glycoproteins of the various virus groups may play a greater or lesser role in assembly and maintenance of the viral envelope structure. however, glycoproteins are essential components for viral infectivity even where they constitute only a minor fraction of the mass of the virion, since they are necessary for the initial events in the viral replication cycle. the fine structure, biological functions, and orgapization of glycoproteins in the viral envelope are discussed in detail in section iv. the carbohydrates of viral glycoproteins are specified in large part by host cell transferases, whereas the amino acid sequences are coded b y the viral genome. glycoproteins of the same virus may exhibit host cell-dependent differences in electrophoretic mobility, which are due to differences in the carbohydrate components (haslam et al., 1970; compans et al., 1970; schulze, 1970) . thus viral glycoproteins may be useful probes to detect differences in glycosylation among various cell types. an important exception to the general finding of host cell-specified carbohydrates is found in myxoviruses and paramyxoviruses. glycoproteins of these viruses lack sialic acid (klenk et al., 1970a,b) , which is believed to be a result of the virus-specified neuraminidase incorporated as a structural component of these virions. all enveloped viruses contain lipid as a major structural component, and available evidence indicates that all the lipid is contained in a single bilayer structure which forms the matrix of the limiting membrane of the virion. investigations using biophysical methods including xray diffraction (harrison et al., 1971) and electron spin resonance (landsberger et al., 1971 (landsberger et al., , 1973 landsberger and compans, 1976) indicate that viral lipids are arranged in a bilayer. with electron microscopy, a well-defined unit membrane is observed at the location of the bilayer. the virus derives its lipids from the host cell membrane where virus maturation occurs, which is the plasma membrane in most instances. therefore a given virus can exhibit marked variation in lipid composition when grown in different cell types, which reflects the composition of the host cell plasma membrane in each instance (klenk and choppin, 1969, 1970; quigley et al., 1971) . apart from minor differences in carbohydrates of glycoproteins, virion proteins are indistinguishable when the virus is propagated in a variety of cells; therefore there appears to be little or no determining influence of viral proteins on the composition of the lipid bilayer. since the viral envelope is continuous with the host cell membrane during morphogenesis, and membrane lipids are characterized by a high rate of lateral diffusion in the plane of the membrane, the similarity in lipid composition between viral envelopes and plasma membranes is not surprising. the distribution of various lipid classes on the internal and external sides of the bilayer has recently been investigated in influenza virions using phospholipase digestion and phospholipid exchange proteins (tsai and lenard, 1975; rothman et al., 1976) . the results indicate that lipids are distributed asymmetrically, with the majority of the phosphatidylinositol and about half of the phosphatidylcholine in the external half of the bilayer and most of the phosphatidylethanolamine and phosphatidylserine in the inner half. these distributions are thought to reflect a similar distribution in the host cell plasma membrane. since less than half of the total phospholipids were accessible b y either approach, it was suggested that glycolipids constitute a significant part of the outer monolayer of the viral membrane. the presence of glycolipids in various enveloped viruses has been demonstrated by chemical analyses , and the types of glycolipids present also reflect those of the host cell. myxoand paramyxoviruses lack neuraminic acid residues in their glycolipids, which again is a likely result of their neuraminidase activity klenk et al., 1970b) . the reactivity of viral glycolipids toward specific lectins demonstrates that their carbohydrates are exposed on the external surface of the bilayer (klenk et al., 1972a) . all polypeptides located internal to the viral lipid bilayer are devoid of carbohydrate, and they may be subdivided into several types according to their structural location and functions. all enveloped viruses possess at least one polypeptide species closely associated with the nucleic acid to form a nucleocapsid or core structure, which is termed the nucleocapsid protein. in addition, viruses with helical nucleocapsids possess another major internal protein, termed the membrane protein, that appears to be associated with the internal surface of the lipid bilayer. most nucleocapsid proteins are not directly involved in interaction with the lipid bilayer, although the nucleocapsids of togaviruses are roughly spherical structures that appear to be closely apposed to the inner surface of the bilayer. the internal membrane (m) proteins of myxo-, paramyxo-, and rhabdoviruses occupy a similar location in the viral envelope. it appears that these proteins are primarily responsible for conferring considerable rigidity on the viral envelope, as compared to that of host cell membranes of similar lipid composition (stoeffel and bister, 1975; landsberger and compans, 1976; lenard et al., 1976) . more complex viruses possess additional internal polypeptides as major components, but their precise locations in the virion have not been established. in addition, minor internal protein components are frequently observed in virions which possess enzymic functions, such as transcriptase activity. such minor polypeptides are not thought to play a direct role in the structure or assembly of the viral envelope. it is well established that carbohydrate chains are covalently linked to glycoproteins and glycolipids of the viral envelope, and both of these components were described above. however, it is uncertain whether or not other complex carbohydrates serve a function in viral envelope structure. the presence of sulfated mucopolysaccharides in highly purified virus preparations of several major groups was recently observed by radiolabeling virions with sulfate pinter and compans, 1975) . these sulfated components are derived from the host cell, since they can be labeled selectively by the growth of cells in 35s0,-containing medium prior to virus infection. in the case of influenza virus, it is possible that these components represent a host cell antigen described to be associated with purified virus particles (knight, 1944) . labeled sulfated mucopolysaccharides can be removed from virus preparations by digestion with hyaluronidase or trypsin without any effect on viral infectivity, suggesting that these components are not essential . it is likely that they associate with the external surface of the virion during the process of maturation. enveloped viruses share many common features in the organization of their structural components, as indicated by several approaches which include electron microscopy, surface-labeling and proteolytic digestion experiments, and isolation of subviral components. much of the evidence pertaining to the arrangement of membrane components has been discussed in other reviews cited above and is only briefly summarized in this section. the organization of the viral membrane corresponds in many respects to the fluid mosaic membrane model . viral glycoproteins appear to be integral membrane proteins which are exposed on the external surface of a lipid bilayer. the external location of the glycoproteins is indicated by their sensitivity to proteolytic digestion and reactivity toward surface-labeling reagents (see review in lenard and compans, 1974) . partial penetration of the glycoproteins into the bilayer is suggested by the fact that segments of the glycoproteins of several virus groups are found to be resistant to protease (gahmberg et al., 1972; mudd, 1974, schloemer and wagner, 1975b; lenard et al., 1976) . in contrast to the glycoproteins, the carbohydrate-free polypeptides of enveloped viruses are located internally to the lipid bilayer, as indicated by their resistance to protease treatment and lack of reactivity toward surface-labeling reagents. these internal proteins are also not reactive toward specific antibodies without disruption of the viral envelope. the best examples of nonglycosylated proteins which appear to be intimately associated with the internal surface of the envelope are the m proteins of myxo-, paramyxo-, and rhabdoviruses. however, whether these polypeptides associate with the bilayer as integral membrane proteins, or as peripheral membrane proteins such as spectrin in erythrocyte membranes, has not been clearly established. in fig. 1 the schematic cross section of an influenza virion shows the general arrangement of its structural components. the extent of penetration of the glycoproteins into the bilayer is not indicated but, fig. 1. schematic cross section of an influenza virion, depicting the arrangement of the major structural polypeptides. the rodlike ha spikes and the more complex na spikes are composed of ha and na polypeptides, respectively. these glycoproteins are exposed at the external surface of the lipid bilayer, and segments may penetrate the bilayer (not shown). the internal membrane-associated m protein appears to form a closely packed layer beneath the bilayer. the helical nucleocapsids are found as multiple discrete segments, each containing an rna molecule coated with a major nucleocapsid protein, (np). the nucleocapsids probably interact directly with the m protein during virion assembly. (from compans and choppin, 1975.) as discussed in section iv, it is likely that a hydrophobic segment penetrates into the bilayer and may even traverse it. a closely packed layer of m protein is depicted on the internal surface of the bilayer in accordance with available information, and the helical nucleocapsids are depicted as twisted hairpinlike structures . it is likely that the nucleocapsids interact with the m proteins during assembly and in so doing participate in determining virion size and shape. little is known about these interactions, and they are not indicated in fig. 1 . in this section we summarize the available information on the detailed structure of the glycoproteins of four virus groups. the infonnation obtained includes the size and shape of viral glycoproteins, the number of polypeptide chains in the complete glycoprotein structure, and compositional data on the polypeptide and oligosaccharide portions of the molecules. detailed structural information on glycoproteins has been obtained for only a few virus types and is obviously incomplete even for the best studied viral systems. influenza viruses possess two glycoproteins with distinct biochemical and morphological properties: hemaggluti-nin (ha) and neuraminidase (na). the ha of influenza virus iii one of the best studied viral glycoproteins. electron microscope studies of the isolated spike structure have shown that it is a rod-shaped triangular prism approximately 14 nm long and 5 nm wide (laver and valentine, 1969) . the ha spike projects radially from the virion envelope, and it is probably a trimer having a mw of about 225,000 daltons (schulze, 1975; wiley et al., 1977) . ha glycoproteins can exist in one of two alternative forms: a single polypeptide with a mw of 75,000 daltons designated ha, or two polypeptides with mws of approximately 50,000 and 25,000 daltons designated ha1 and ha2, respectively. ha, and ha, are proteolytic cleavage products of ha, which remain cross-linked by disulfide bonds (lazarowitz et al., 1971; laver, 1971) . the extent of cleavage of ha to ha, and ha2 may vary from none to complete cleavage of all ha polypeptides (lazarowitz et al., 1973) . the extent of cleavage de-pends upon several factors including the host cell, the virus strain, and the presence or absence of proteases in the culture medium. the cleavage of ha may not be a requirement for infectivity of all virus strains, and spike morphology is not altered detectably by cleavage of ha to ha, and ha,, but recent studies have shown that cleavage enhances the infectivity of some influenza virus strains (klenk et al., 1975 ; lazarowitz and choppin, 1975) . the tryptic peptide maps of ha, and ha, are distinct, and the only notable difference in the amino acid composition of the two polypeptides is the proline content (laver, 1971) . the proline content of ha, is at least six times higher than that of ha,. ha, is tightly folded on itself, probably reflecting the high proline content. a portion of the ha spike is released by protease treatment as a soluble protein which can be crystallized (brand and skehel, 1972) . functional ha spikes have been isolated from several influenza strains by treating virions with ionic or nonionic detergents. after removal of the detergent, the spikes aggregate, forming rosettelike structures, suggesting that the base of the ha spike is hydrophobic in nature (laver and valentine, 1969) . available evidence indicates that the ha spike is amphipathic and that a portion of it is buried within the lipid bilayer of the viral envelope. this is indicated b y the finding that protease-treated virions retained ha, or portions thereof while having no identifiable spikes (compans et nl., 1970; lenard et al., 1976) . the ha, glycoprotein therefore contains the hydrophobic end of the spike. this glycoprotein aggregates even in the presence of guanidine hydrochloride (laver, 1971) . the composition of a 50-residue peptide associated with the lipid bilayer, which is removed from the carboxyl terminus of ha2 by bromelain, has been estimated, and 21 of the amino acids are hydrophobic in nature, whereas 14 may be charged (skehel and waterfield, 1975) . the carboxyl terminus of this peptide is thought to be buried within the lipid bilayer. the aminoterminal end of ha, seems to extend radially from the virion surface, and a sequence of 10 residues from the amino-terminal end of ha, is highly conserved in both type-a and type-b influenza virions (skehel and waterfield, 1975) . the hydrophobicity of ha, suggests that it is similar to other membrane-associated glycoprotein segments (segrest et al., 1972; nakashima et al., 1975) , but freeze-fracture studies of the influenza virion indicate that extensive portions of ha and na do not penetrate through the lipid bilayer (bachi et al., 1969) . circular dichroism studies of ha spikes isolated intact and those cleaved from the virion surface may be useful in further defining the secondary structure of the hydrophobic regions. until recently, little information was available concerning the carbohydrate portion of the ha glycoprotein. about 80% of the carbohydrate attached to viral proteins was shown to be linked to ha; the gly-cosy1 moieties were shown to be comprised of glucosamine, mannose, galactose and fucose at a molar ratio of 6 :4 : 1 : 1, respectively, and the total carbohydrate of ha, determined by chemical means, was estimated to be approximately 12,000 daltons (laver, 1971) . it was also found that ha, of influenza a virions contains fucose, whereas ha, of influenza b (gl1760) does not . recently the glycopeptides obtained by pronase digestion of influenza a viruses have been characterized (schwarz et al., 1977; nakamura and compans, 1978b) . both groups showed that ha possesses type i (oligosaccharide side-chain comprised of glucosamine, mannose, galactose, fucose and sialic acid) and type i1 (oligosaccharides comprised of glucosamine and mannose) glycopeptides, but the distribution of each type on ha, and ha, was shown to depend upon the virus strain. schwarz et al. (1977) showed that ha, of two avian influenza viruses possessed only type i glycopeptides, whereas both type i and i1 glycopeptides were shown to be present on ha,. however, nakamura and compans (197813) using the wsn strain of influenza showed that ha, possessed both type i and i1 glycopeptides whereas ha, contained only type i glycopeptides. since it is likely that ha, occupies a similar structural location in wsn and avian hemagglutinin proteins, these results suggest that specific amino acid sequences determine whether a type i or type i1 oligosaccharide is added at a particular site on the glycoprotein. the host cell type may also determine the type of oligosaccharide chains added to a given viral glycoprotein. thus, although ha, of wsn strain influenza virions grown in mdbk cells contained only type i glycopeptides, both type i and i1 glycopeptides were found in ha, when grown in cef cells (nakamura and compans, 1978~) . schwarz et al. (1977) showed that the type i ha glycopeptides of fowl plague and n influenza strains grown in chicken embryo fibroblasts (cef) had a molecular weight of approximately 2,600 daltons, whereas type i1 glycopeptides from the same source has a molecular weight of approximately 2,000 daltons. the molecular weights of type i and i1 glycopeptides of the wsn strain of influenza virus were also found to have approximately this size (nakamura and compans, 1978b) . ha glycopeptides of virus grown in mdbk cells were slightly larger in size than those of virus grown in cef cells. based upon the estimated carbohydrate content (12,000 daltons) of the ha glycoprotein obtained by laver (1971) and schwarz and klenk (1974) and the size estimates of the type i and i1 glycopeptides of influenza virus grown in mdbk cells, it was estimated that ha, contains a single type i glycopeptide whereas ha, possesses two type i and one or two type i1 oligosaccharide side-chains for the wsn strain (nakamura and com pans, 1978b) . the na spike, as shown in fig. 1 , also projects radially from the lipid bilayer of the influenza virion but is morphologically distinguishable from the ha spike (laver and valentine, 1969) . the na spike is elongated and has a square knoblike structure at one end. the oblong head is approximately 5 x 8.5 nm, and it is attached to a fiber approximately 10 nm long. the na spike has a mw of approximately 240,000 daltons and is comprised of four na polypeptides each having a mw of approximately 58,000 daltons; two na polypeptides appear to be linked by disulfide bonds to form dimers, which are thought in turn to aggregate by noncovalent bonds to form the tetrameric spike (bucher and kilbourne, 1972; lazdins et al., 1972) . the amino acid composition of the na has been determined, and it has a cysteine content significantly higher than that of the other viral polypeptides (laver and baker, 1972) . influenza na is in some ways morphologically similar to enzymes involved in sugar metabolism in the gut, as discussed by forstner and riordan in this volume. after trypsin treatment a tetramer of four coplaner subunits of 4 x 4 x 4 nm was isolated (wrigley et al., 1973) . this structure appears to correspond to the knob, and it is enzymically active. the knob structure can no longer aggregate with itself or with ha molecules, indicating that the hydrophobic regions of the molecule have been removed by proteolysis. the mw of the portion of the na monomer which remains associated with the envelope after trypsin treatment was estimated to be 7000 daltons by lazdins et al. (1972) and 12,000 daltons by wrigley et al. (1973) . presumably, this portion functions to attach the na molecule to the virion and plays no role in the enzymic properties. furthermore, the portion of the spike which remains associated with the viral envelope is more highly glycosylated than the knob-shaped portion of the na spike with enzymic activity (lazdins et al., 1972) . na is present in influenza virions in smaller amounts than ha, in a ratio of about three to four ha polypeptides for each na polypeptide. little information is available on the carbohydrate components of the na. recently, both the ha and na of influenza virions were shown to be sulfated glycoproteins . the sulfate appears to be covalently linked to the oligosaccharide chains of viral gly-coproteins compans, 1977, 1978a) . glycoproteins of enveloped viruses of all the other major groups studied also are sulfated, whereas carbohydrate-free polypeptides are not kaplan and ben-porat, 1976 ). rhabdoviruses are covered with closely spaced glycoprotein spikes approximately 10 nm in length as shown by electron microscope studies. rhabdoviruses possess only a single glycoprotein species termed the g protein, and for vesicular stomatitis virus (vsv) it has a mw of approximately 69,000 daltons (wagner, 1975) . carhvright et d . (1972) have postulated that only a single glycoprotein molecule constitutes the spike structure. the g proteins of rhabdoviruses are amphipathic, like the glycoproteins of influenza virions. after protease treatment of vsv a fragment of the g protein was demonstrated to be associated with the intact virion (mudd, 1974) . more recently, schloemer and wagner (1975a) isolated a small nonglycosylated portion of the g protein from the envelope of protease-treated vsv virions. the fragment was found to have a mw of 5200 daltons, approximately equivalent to 50 amino acids. this is similar to the estimated size of the portion of the ha2 protein of the influenza virion thought to be buried within the lipid bilayer. amino acid analysis of the g-protein fragment showed that it contained a preponderance of hydrophobic amino acids. as is the case for the membrane-associated portions of influenza ha, the hydrophobic fragment of the g protein is long enough to penedate the lipid bilayer. conclusive evidence for such penetration has not been obtained, but cross-linking experiments with glutaraldehyde suggest that interactions may occur between g proteins and internal m proteins (brown et d . , 1974) . the carbohydrates linked to the g protein of vsv have been studied in detail. the glycoprotein is 9-10% carbohydrate by weight and contains mannose, galactose, n-acetylglucosamine, and neuraminic acid as the major sugar components, with lesser amounts of n-acetylgalactosamine and fucose (mcsharry and wagner, 1971 ; huang, 1970. etchison and holland, 1974a) . the size and composition of the carbohydrate moieties per vsv glycoprotein are variable, depending upon the cell type in which the virions are grown (burge and huang, 1970; etchison and holland, 1974a) . likewise, the sequence of the carbohydrates within the glycosyl side-chains may exhibit cell depen-dence (moyer and summers, 1974) . the monosaccharide composition of the oligosaccharide side-chains is similar to that of influenza virus (etchison and holland, 1974b) , with the exception that sialic acid is present on the termini. klenk et az. (1970b) demonstrated the presence of sialic acid on the envelope of vs virions by treating the virus with colloidal iron hydroxide which stains sialic acid residues. the stain was shown to bind to the envelope of vsv, whereas it did not bind to influenza virions or paramyxoviruses as shown by electron microscopy. more recent studies have shown that the glycosyl moieties are not terminated by sialic acid when vsv is grown in mosquito cells, because these cells lack sialyl transferase (schloemer and wagner, 1975b) . preliminary studies indicated that from 3-5 cyanogen bromide peptides of the vsv g-protein may be glycosylated (wagner, 1975) . however, etchison and holland (1974a,b) have calculated that there are only 2 glycopeptides of 3000-3400 daltons in each g protein molecule. more recent studies by etchison et al. (1977) have indicated that the vsv g-protein possesses two identical glycopeptides. the number average molecular weight of the glycopeptides was estimated to be 3150 by gel filtration analysis and 3450 based on composition of the amino acid and sugar residues. additional information concerning the structure of the glycopeptide was obtained by sequential chemical and enzymatic degradation. these results indicate that the glycopeptides are acidic type i glycopeptides with two or three mannose branches terminating in sialic acid. moyer and co-workers (1976) have obtained evidence that the glycosyl residues are covalently linked to an asparagine residue of the g protein, and the sequence of the monosaccharides that constitute the oligosaccharide side-chains has been determined (hunt and summers, 1976b) . togavinises are small, spherical enveloped viruses 50-60 nm in diameter with a core structure that appears to be icosahedral. although there are many members of the togavirus group, the best studied are sindbis virus and semliki forest virus (sfv). sindbis virus possesses two glycoproteins designated el and e,; both have a mw of approximately 50,000 daltons, and they are not linked by disulfide bonds because they can be separated under nonreducing conditions and without alkylation (schlesingeret al., 1972) . sfv is similar to sindbis virus in that it also has 2 glycoproteins of similar molecular weight designated el and e2, but a third glycoprotein designated e, has also been detected. the molecular weights of el, e,, and e, were estimated to be 49,000,52,000 and 10,000 daltons, respectively (garoffet al., 1974) . e, and e, are synthesized as a common precursor protein (nvp68) that is cleaved to yield e, and e,. el, ep, and e, of sfv are present in equimolar ratios as are e, and e2 of sindbis virus. the arrangement and relationship of the glycoproteins within the spike structure of the sindbis and sfv virion are yet to be resolved. and garoff (1974) have used the cross-linking agent dimethyl suberimidate to study the interrelationships of the glycoproteins of sfv. el and e, were most readily cross-linked, but steric hindrance may have prevented the cross-linking of el, e2, and e,. recent studies by jones et al. (1977) have suggested that el and e, of sindbis virus, like el, ez, and e, of sfv, probably constitute the glycoprotein spike, since the cleavage of pep, a precursor of e,, does not occur in temperature sensitive mutants of complementation groups including that thought to represent el. furthermore, cleavage is inhibited by antibodies directed against either el or e,. these data suggest that pe, and el may exist as a complex in the membrane of the infected cell, and presumably also that el and e, remain as a complex in the viral envelope. protease treatment of sfv cleaves the glycoproteins, el and e,, and residual segments can be isolated from the envelope of spikeless particles (utermann and simons, 1974) . the amino acid composition of each of these peptides demonstrates that they are enriched in hydrophobic amino acids. the residual peptides have a mw of approximately 5000 daltons; hence they are comprised of about 50 amino acids. thus, like the glycoproteins of influenza virus and vsv, togavirus glycoproteins are amphipathic in nature and appear to possess a peptide of similar size embedded within the viral envelope. when sfv was treated with high concentrations of dimethyl suberimidate, both the tail fragments of el and ez were cross-linked with the nucleocapsid protein, which supports the conclusion that the hydrophobic segments, of el and e, may penetrate through the lipid bilayer and interact with the nucleocapsid . carbohydrate analysis of sfv showed that el contains about 18 moles of monosaccharide, e2 about 28 moles and e, about 22 moles . e2 seemed to be particularly rich in mannose. it has recently been reported that the sfv glycoproteins el, e,, and e, are differentially glycosylated (mattila et al., 1976) . el and e, were shown to contain, on the average, one type a glycosyl side chain. e2 was shown to contain one type a glycosyl side chain and possibly one or two b-type glycosyl side chains. a-type oligosaccharides are complex structures containing fucose, galactose, mannose, and n-acetyl-glucosamine, whereas b-type oligosaccharides contain only mannose and n-acetylglucosamine (johnson and clamp, 1971 (1975) showed that one type-a and one type-b residue were attached to each of the two different glycoproteins. the extent of completion of the glycosyl side chains was shown to be dependent in part upon the cell in which the virus was grown (burge and huang, 1970; keegstra et ul., 1975) . bhk-21 cells were able to complete type-a side chains (i.e., to add galactose and terminal sialic acid), whereas chicken embryo fibroblasts were quite inefficient in adding sialic acid (keegstra et al., 1975) . both sfv and sindbis virus possess hemagglutinating activity. recent studies by dalrymple et al. (1976) localized this activity to the el glycoprotein of sindbis virus, whereas the e2 glycoprotein possesses the antigenic determinants which react with neutralizing antibody. the virions of paramyxoviruses are covered with glycoprotein surface projections approximately 10 nm in length. two functionally distinct types of glycoproteins have been isolated from several members of the paramyxovirus group (scheid et al., 1972; scheid and choppin, 1973; . in contrast to the situation in influenza viruses, which possess hemagglutinating and neuraminidase activities on distinct glycoprotein molecules, both of these activities are associated with the larger of the two glycoprotein species in paramyxoviruses, which is designated the hn glycoprotein. the smaller glycoprotein component in paramyxoviruses is associated with cell fusion and hemolysis activities and is designated the f glycoprotein. purified glycoproteins of sv5 form rosettelike clusters in the absence of detergents, suggesting that the spikes have hydrophobic bases (scheid et al., 1972) . the aggregates formed are morphologically distinguishable, the hn protein forming berrylike aggregates while the f protein forms distinct rosettelike clusters consisting of radiating spikes 10-13 nm in length with distinct terminal knobs. in the three best studied paramyxoviruses, sv5, newcastle disease virus (ndv), and sendai virus, the h n glycoprotein has a mw range of 65,000-74,000 daltons, and the hn spike solubilized by detergent treatment sediments at 8.9s (scheid et al., 1972) . although the exact size and fine structure of the hn spike remain to be determined, it has been suggested that each morphological spike contains at least two glycoprotein monomers . in some strains of ndv, an 82,000-dalton precursor of the hn glycoprotein designated hno is incorporated into virions . these particles have reduced hemagglutinating and neuraminidase activities which are activated upon proteolytic cleavage, which produces the 74,000 dalton hn molecule. in svs, ndv, and sendai virions, the f glycoprotein has a mw of -56,000 daltons, and the f spike that is solubilized by treatment of sv5 virions with triton x-100 has a sedimentation coefficient of approximately 6.7s (scheid et al., 1972) . in sendai virions (homma and ohuchi, 1973; scheid and choppin, 1974) and certain strains of ndv (nagai et al., 1976) grown in some cell types, the f glycoprotein is not found, and a larger precursor molecule designated fo is observed. recent studies have indicated that proteolytic cleavage of the fo glycoprotein yields two cleavage products, which have bekn designated f1 and f, (shimizu et al., 1974; nagai et al., 1976; scheid and choppin, 1977) . the f2 cleavage product is more highly glycosylated than f1, and appears to be located on the distal end of the spike; it contains a blocked n-terminal as is also found in the uncleaved f, glycoprotein (scheid and choppin, 1977) . thus the cleavage of f, generates a free n-terminal on the f, segment of the glycoprotein, and this appears to expose a new hydrophobic region of the molecule which may be important for virus-induced cell fusion. the f, glycoprotein is inactive in cell fusion and hemolysis and can be converted into the active f glycoprotein by proteolytic cleavage, with concomitant activation of cell fusion and hemolysis. it is of particular interest that virions containing the fo precursor are not infective and gain infectivity upon such proteolytic cleavage. in the case of enveloped viruses, glycoproteins located on the surface of the virion are the components involved in adsorption to cellular receptors, which may or may not be host cell glycoproteins. hemagglutination by influenza virus has been studied as a model system for the adsorption of a virus to receptors, and considerable information has been obtained. adsorption to the erythrocyte occurs by the ha spike binding to sialic acid-containing components on the cell surface. the receptor molecule has been isolated from chick red blood cells, and it is a major glycoprotein containing m and n blood group anti-gens. a detailed summary of the properties of this receptor is given by schulze (1975) , as well as by tanner in this volume. removal of sialic acid from the receptor molecule by na prevents the agglutination of erythrocytes by influenza virus. hemagglutination can also be inhibited by pretreating the virus with specific antibodies. while the model for adsorption of influenza virus to erythrocytes seems straightforward, its applicability to the adsorption of viruses to other cell types is uncertain, and little information is available on the nature of receptors for viruses. liposomes containing gangliosides may be capable of acting as receptors for sendai virus, a paramyxovirus (haywood, 1974 (haywood, , 1975 . sialyoglycoproteins inserted in liposomes can also act as receptors, but the fact that gangliosides can act as receptors raises the possibility that glycolipids may be involved in viral attachment. further evidence that viral glycoproteins specify virus-host cell interactions has been obtained from the studies of bishop et al. (1975) . they produced spikeless particles of the indiana serotype of vsv b y treating the virus with bromelain or pronase. these particles were shown to be noninfectious but, when they were reconstituted with purified g protein isolated from the same strain or from the new jersey serotype, infectivity was restored. antibody directed against the homologous g protein used for reconstitution effectively neutralized the virus, but antibody directed against the serotype of the spikeless particle was ineffective in neutralization when glycoproteins from a different serotype were used for reconstitution. the mechanism of adsorption of other enveloped viruses has not been studied to the same degree as that of ortho-and paramyxoviruses. herpes viruses do not exhibit hemagglutinating activity, but they possess at least 13 glycoproteins which are asymmetrically located on the external surface of the envelope (roizman and furlong, 1974; o'callaghan and randall, 1976) . removal of the envelope by nonionic detergents irreversibly alters the infectivity of these viruses (abodeely et al., 1970) . oncornavinis glycoproteins react specifically with host cell receptors and in the case of avian leukosis viruses they define the host range as well as the classification into subgroups based on interference and neutralization properties ishizaki and vogt, 1966; duff and vogt, 1969) . in addition, tozawa et al. (1970) showed that the viral glycoproteins absorbed homologous neutralizing antibody but not antisera prepared against heterologous virions. the purified glycoproteins were shown to interfere with the early steps in infection by the homologous virus. the determination of host range by the glycoproteins of avian leukosis viruses was further demonstrated by phenotypic mixing experiments. defective rous sarcoma virions that lack the envelope gene cannot infect cells. however, when grown in the presence of an avian leukosis virus, they acquire the glycoproteins of that virus, and the host range of the sarcoma virus reflects that of the leukosis virus (hanafusa, 1965; vogt, 1965 ; kawai and hanafusa, 1973) . the major glycoprotein of rauscher murine leukemia virus (gp71) binds specifically to receptor molecules found on murine cells but not on other mammalian cells (delarco and todaro, 1976). the phenomenon of cell fusion may be caused by members of several groups of enveloped viruses of which paramyxoviruses are the best studied. the properties of hemolysis and cell fusion are associated with the f glycoprotein as described above. virus-induced cell fusion may occur in the absence of virus replication, in the presence of high concentrations of virus particles. both infectious and noninfectious viruses are equally adept at causing such cell fusion which sometimes has been termed fusion from without (bratt and gallaher, 1972) . in contrast, fusion with low multiplicities of virus has been called fusion from within and is dependent upon replication of the virus. it is likely that the f glycoprotein is involved in both types of fusion phenomena. the process of fusion through the direct action of concentrated virus has been studied intensively since it was first observed by okada (1958) . sendai virus-induced fusion involves several separate identifiable steps, as indicated by maeda et al. (1977) : (1) adsorption of the virus to the cell, which seems to occur at the tips of the spikes located on the virion surface; (2) aggregation of cells; (3) fusion of the viral envelope with the cell membrane and finally fusion of the cells. these studies suggest that the envelope bilayer of the virion and the membrane of the target cell are brought into contact by the action of the viral hn glycoprotein. the f protein then causes destabilization of the lipids, and an intermixing of lipids occurs. the mechanism of action of the f protein remains to be determined. a neuraminidase activity has been shown to be associated with orthomyxoviruses and paramyxoviruses. the functional role of this enzyme was uncertain for many years, and there was even doubt at some point that it was a viral gene product, since similar enzyme ac-tivity is present in normal cells (white, 1974); however, these doubts have been resolved by studies of the biochemical, genetic, and antigenic properties of viral neuraminidase (see review in bucher and palese, 1975) . several distinct functions have been proposed for this enzyme. it is postulated that neuraminidase-containing virus lodges in the upper respiratory tract and binds to mucin via ha. the glycosyl residues of muchin are terminated by sialic acid (n-acetylneuraminic acid) to which ha binds, and one role of the neuraminidase may be to cleave the sialoglycoprotein bond, freeing the bound virion. it is presumed that in this way the virus is released, and that underlying cell receptors are exposed to which ha can attach (davenport, 1976 ). it has also been postulated that neuraminidase is involved in an early event such as penetration, but this is unlikely because virions remain infectious after inhibition of neuraminidase activity by specific antibody (bucher and palese, 1975) . however, the possibility that neuraminidase participates in release of the budding virion from the infected cell surface has gained support from several types of experiments. by using influenza strains with different levels of enzyme activity, it was shown that virus strains having low activity were released from cells more slowly than strains with higher enzyme activity (palese and schulman, 1974) . further, in the presence of antibody to viral neuraminidase, which inhibited enzyme activity, virions were formed but release of virus into culture media was inhibited (seto and rott, 1966; compans et al., 1969; webster, 1970 more conclusive information on the function of the enzyme has been obtained with temperature-sensitive mutants of influenza virus which are defective in neuraminidase activity . at the nonpermissive temperature, no neuraminidase activity is detected; virus particles are produced by cells, despite the fact that infectivity titers are markedly reduced. however, the virus particles form large aggregates, and in contrast to wild-type virions these particles contain sialic acid as shown by colloidal iron hydroxide staining. since influenza virions bind to sialic acid residues, these results indicate that the mutant virus particles aggregate to each other, because sialic acid is added to viral carbohydrates, and that the essential function of viral neuraminidase is to remove or prevent the addition of such sialic acid. in support of this conclusion, the addition of bacterial neur-aminidase to cells infected with these mutants cause a marked enhancement of virus release. further evidence supporting this role for the neuraminidase was obtained with a neuraminidase inhibitor, 2-deoxy-2,3-dehydro-ntrifluoracetylneuraminic acid (fana). influenza virus grown in the presence of fana contains neuraminic acid on its envelope, and the particles undergo extensive aggregation (palese and compans, 1976) . a marked reduction in virus yield is observed because of this aggregation, and treatment with purified neuraminidase results in a marked enhancement in progeny virus yields, apparently through disaggregation of virus. thus viral neuraminidase is not required for assembly of progeny virions but appears to be essential for the removal of sialic acid from the surface of the virion itself. from the studies described above it is evident that the membranes of enveloped viruses are asymmetrically constructed, with the glycoproteins that comprise the spikes or surface projections physically located on the exterior of the viral envelope. thus the glycoproteins are exposed to the immune surveillance system of the host and, being good immunogens, may elicit humoral and cellular immune responses (evans, 1976) . the classification of virus isolates into specific strains depends largely upon serological procedures, and for enveloped viruses, surface glycoproteins are usually the relevant antigens in such tests. neutralizing antibodies are usually directed against the viral proteins involved in attachment to receptors, e.g., ha glycoprotein of influenza virus (webster and laver, 1975) , g protein of vsv (wagner, 1975) , gp69/71 of murine leukemia viruses (fischinger et az., 1976; strand and august, 1976) , gp85 of avian leukosis viruses (bolognesi, 1974) , and e, of sindbis virus (dalrymple et d., 1976) . high concentrations of antibody can prevent attachment of the virus to receptors, but low concentrations can also neutralize infectivity by a mechanism that is not understood. the specific determinants of viral glycoproteins recognized by virus-neutralizing antibodies have not been chemically characterized. it has been postulated that antibody molecules may recognize only determinants on the tip of the ha spike of the influenza virion (white, 1974) . in reaching these conclusions, it has been assumed that the size of the antibody molecule precludes the possibility that it could make contact with any other part of the spike, since the spaces between the spikes are too small for the immu'noglobulin to interact with other regions. electron microscopic observations (lderty and oertilis, 1963) indicate that antibody molecules interact with the tips of surface spikes, and analysis of the tryptic peptides of ha molecules isolated from *closely related influenza strains has shown that they rarely differ b y more than one or two peptides webster and laver, 1972) , suggesting that the strain differences are indeed restricted to small regions or determinants of the ha spike. the antigenic character of many viral glycoproteins is stable, but the determinants exhibited by the glycoproteins of influenza virions are characteristically variable, as evidenced by the many different strains of type-a and -b influenza. the ha and na glycoproteins of influenza viruses are antigenically distinct, and they undergo antigenic changes independently of each other. the antigenic changes that occur may be gradual, in which case the different virus strains are clearly related to each other with respect to both surface antigens. antigenic changes of this nature are termed antigenic drift, and they result from the interplay of viral mutability and immunological selection (webster and laver, 1975) . the presence of antibody of low avidity may select for single-step mutants, which have an altered amino acid in the key area of the antigenic determinant, giving rise to a new viral strain. at intervals of 10-15 years sudden and complete changes in the determinants of type-a influenza glycoproteins occur; the changes are such that the viruses that arise possess glycoproteins that appear completely distinct on peptide mapping . dramatic changes in the antigenic determinants of viral glycoproteins are termed antigenic shifts, and it is these new viruses that cause worldwide influenza pandemics. it has been postulated that antigenic shift may occur because type-a strains of human origin may undergo recombination with type-a strains of avian and animal origin. the antigenic determinants of the new ha or na are sufficiently different that the human host does not possess immunity, and the virus gains a selective advantage. interestingly type-b influenza viruses do not undergo antigenic shift. the reason for this may lie in the fact that type-b influenza viruses have not been isolated from other animal species; thus it is probable that the type-b viruses can not undergo similar recombination events (webster and laver, 1975) . viral glycoproteins provide excellent systems for analysis of the function of carbohydrates in membrane glycoproteins. several approaches have been used to modify the carbohydrates, including treatment of virions with specific glycosidases, growth of virus in cells with specific sugar transferase defects, and treatment of virus-infected cells with inhibitors of glycosylation. removal of sialic acid from the g protein of vsv has been reported to reduce the infectivity of vsv virions (schloemer and wagner, 1974) , whereas the infectivity of sfv (kennedy, 1974) and friend leukemia virus (schafer et al., 1977) were reportedly unaltered by such treatment. moreover, enzymic addition of sialic acid to the glycoproteins of sindbis virus did not alter the infectivity of this virus (stollar et al., 1976) , while enhancing the infectivity of influenza virus (schulze, 1975) and restoring infectivity to neuraminidase-treated vsv (schloemer and wagner, 1974) . treatment of influenza virions with glycosidases alter hemagglutinating activity, but neuraminidase activity was unaffected; however, similar treatment of ndv, a paramyxovirus, resulted in no alteration in hemagglutinating or neuraminidase activities (bike1 and knight, 1972) . schiifer et al. (1977) showed that the indirect hemagglutinating activity of friend leukemia virus was inhibited by glycosidase treatment but that viral infectivity and determinants involved in viral interference and absorption of neutralizing antibody were unaltered. sindbis virus were grown in these cells, the apparent mws of their glycoproteins were lower. the infectivity of vsv and sindbis virus grown in these cells was not altered from that of fully glycosylated virions. however, glycosidase treatment of sfv was shown to decrease the infectivity of this virus (kennedy, 1974) . the apparent difference between these results remains to be resolved. it is possible that the core of the glycosyl side chain is added to the sindbis virus glycoproteins in the enzyme-deficient cells, whereas the glycosidase treatment used by kennedy may have removed more of the glycosyl moieties from the sfv glycoproteins. inhibitors of glycosylation have also been used to assess the role of glycosyl side chains of glycoproteins. primarily, three different inhibitors have been used for this purpose: 2-deoxy-~-glucose (2-dg), an analog of glucose which substitutes for mannose, preventing the further addition of monosaccharides to the glycosyl side chain; dglucosamine, which is thought to inhibit glycosylation at high concentrations by decreasing utp pools in the cell and subsequent activation of other sugars (scholtissek, 1971) ; and tunicamycin (tm), a glucosamine-containing antibiotic that inhibits the formation of n-acetyl-glucosamine-lipid intermediates which serve as donors for the synthesis of the oligosaccharide side chains of glycoproteins (tkacz and lampen, 1975) . kilbourne (1959) and kaluza et al. (1972) were among the first investigators to utilize inhibitors of glycosylation to study the role of carbohydrates in influenza virions. they showed that 2-dg and d-glucosamine inhibited the biosynthesis of active ha, na, and mature infectious influenza virions. subsequent biochemical studies revealed that high concentrations of 2-dg or dglucosamine prevented the synthesis of influenza virus glycoproteins (klenk et al., 1972b nakamura and compans, 1978a) . instead, an unglycosylated or incompletely glycosylated hemagglutinin precursor ha,, was detected. once synthesized, hao was found associated with cytoplasmic membranes, as is the case with the normal glycoprotein. hao glycoprotein in cells infected with the fowl plague strain (fpv) was cleaved by cellular proteases to yield a heterogeneous product . however, the comparable protein synthesized in cells infected with the wsn strain was processed and incorporated into virions (nakamura and compans, 1978a) . because 2-dg and d-ghcosamine interfere with metabolic reactions other than the glycosylation of glycoproteins, they may cause side effects that can affect virus replication. therefore tm, a compound that seems to affect only the glycosylation of glycoproteins, has been employed to extend these studies. t m inhibited virion formation in fpvinfected cells and the unglycosylated glycoprotein ha,, appeared to be degraded by cellular proteases (r. t. ; ha,, of the wsn strain synthesized in the presence of tm also appeared to be completely unglycosylated, whereas even at high concentrations of 2-dg and dglucosamine some glycosylation occurred (nakamura and compans, 1978a) . nonetheless, tm did not inhibit virion formation to the same extent as 2-dg (nakamura and compans, 1978) . the surface spike layer of wsn virions produced in the presence of 2-dg, d-glucosamine, and tm was altered morphologically, and the hemagglutinating activity of the virions was significantly reduced. these results suggest that glycosylation of virion glycoproteins is not required for influenza virion formation but is needed for biological activity of viral glycoproteins. these glycosylation inhibitors have also been used with several other enveloped viruses. when herpes viruses are grown in the presence of 2-dg, the infectious virus yield is decreased by greater than 95%, but the yield of viral particles is not reduced (courtney et al., 1973) . the reduction in infectivity was attributed to an inability of the virions to attach to the host cell and penetrate, implying that oligosac-charide residues of herpes virus glycoproteins play a role in the attachment and recognition of host cell receptors. the glycoproteins of vsv, sindbis virus, and sfv are not glycosylated in the presence of tm (r. t. leavittet al., 1977) . mature vsv and sindbis virions are not released from tmtreated cells, but nonglycosylated precursors are synthesized and seem to be stable within the cell (leavitt et al., 1977) . similarly, r. t. schwarz et al. (1976) showed that unglycosylated sfv glycoprotein precursors were synthesized in tm-treated cells. moreover, they were not degraded by host cell proteases and virion assembly was completely inhibited. it is interesting that, as noted above, ha, of fpv is degraded when it is grown in tm-treated chicken embryo fibroblasts, but the glycoprotein precursors of sfv synthesized in the same cells are stable. these results may be due to greater release of protease by fpv infection or, alternatively, the polypeptide backbone of sfv glycoproteins may be less susceptible to proteolytic degradation. high concentrations of glucosamine rapidly shut off the production of infectious avian sarcoma virus particles (hunter et al., 1974) . however, avian sarcoma virus particles were assembled and released at about 60% of the control level in tm-treated cells, and such particles appeared to lack glycoproteins (r. t. . the infectivity titer of the virus produced in tm-treated cells decreased by only 15%. these results indicate that the effects of glycosylation inhibitors may vary with the virus and host cell, as well as with the specific inhibitor used. side effects of some inhibitors may have marked effects on virus replication. however, the fact that in some systems virus particles are produced with unglycosylated or incompletely glycosylated glycoproteins clearly demonstrates that the complete glycosylation process is not essential for intracellular migration of glycoproteins or their incorporation into the plasma membrane and subsequently into virus particles. the biological activities of some glycoproteins such as those of influenza virus and sfv appear to require glycosyl moieties, whereas for other viruses, such as murine leukemia virus, this may not be the case. the membranes of enveloped viruses, particularly those of singlestranded rna viruses, are unique in their simplicity of construction and are useful systems for studying the interactions of specific proteins with the lipid bilayer. hence comparative studies of the effects of viral proteins of the lipid bilayer structure have been made using the techniques of electron spin resonance (esr), nuclear magnetic resonance (nmr), and fluorescence polarization (fp). esr studies have provided evidence that the lipids of influenza virus, sv5, rauscher leukemia virus, vsv, and sfv (landsberger et uz., 1971 (landsberger et uz., , 1972 (landsberger et uz., , 1973 ; sefton and gaffney, 1974) are bilayer structures with fluid lipid phases similar to those observed for other biological membranes, but all viral membranes were shown to be substantially more rigid than the corresponding host cell plasma membrane. proteolytic removal of the glycoprotein spikes from the surface of sv5 and influenza virions did not appreciably alter the fluidity of the lipid bilayer of these viruses. lenard et al. (1976) have provided further evidence that the glycoproteins of the influenza virion contribute little to the rigidity of the lipid bilayer. the phospholipid composition of standard influenza particles, and "incomplete" virus produced upon serial undiluted passage, were compared and found to be indistinguishable, as were the esr spectra of the two types of particles. the incomplete particles were shown to contain approximately twice the amount of glycoproteins relative to the complete particles. thus it was concluded that the rigidity of viral membranes may be determined b y the m protein and not by the viral glycoproteins. this may not be entirely the case for vsv and sfv, since sefton and gaffney (1974) and landsberger and compans (1976) observed that, when the glycoproteins of these viruses were removed by proteases, the envelope became more fluid, indicating that the viral glycoproteins may contribute to the rigidity of the envelope. however, landsberger and compans (1976) postulated that the major effect on vsv bilayer fluidity was exerted by the m protein, since the fluidity of the lipid bilayer was altered only slightly when the g protein was removed by protease, whereas vesicles prepared from extracted viral lipids were much more fluid than lipids in virions. using the technique of fluorescence depolarization, moore et az. (1976) and barenholz et al. (1976) showed that the envelope of sfv, sindbis virus, and vsv has a higher microviscosity than that of the plasma membranes from which the virions budded. the increased microviscosity was attributed in part to insertion of the hydrophobic regions of the glycoproteins into the envelope bilayer. stoffel and bister (1975) , using nmr spectra of '3c-labeled lipids, also demonstrated that the envelope lipids of vsv are highly rigid, as a result of either lipid-lipid or lipid-protein interactions. in general it may be concluded therefore that the lipid bilayer of enveloped virions is more rigid than the host cell membrane from which the virion buds. while virion glycoproteins do interact with the lipid bilayer and may affect the rigidity of the membrane to some extent, the internal membrane protein(s) may also be of equal or greater importance in determining membrane rigidity. most enveloped viruses form by a process of budding at the plasma membrane, with little or no participation of other membrane structures in the final steps of maturation. however, there are important exceptions in the case of certain virus groups. the capsids of herpes viruses are assembled in the nucleoplasm and are observed to bud through the inner nuclear membrane, acquiring their envelopes in the process (roizman and furlong, 1974; o'callaghan and randall, 1976) . they appear to be transported as enveloped particles through cytoplasmic channels to the cell surface. bunyaviruses (murphy et az., 1973) and coronaviruses (oshiro, 1973) appear to form primarily by budding into cytoplasmic cisternae, and extracellular virions are observed associated with the plasma membrane. for the rhabdovirus group, several modes of maturation have been reported. vsv, the most widely studied member, usually forms by budding at the cell surface, but maturation at intracellular membranes has been observed. the new jersey strain of vsv was observed to bud primarily from plasma membranes of l or vero cells, and almost entirely at intracytoplasmic membranes of pig kidney cells (zee et al., 1970) . these reports indicate that the site of maturation of a specific virus type may vary depending on the host cell. rabies virus, which resembles vsv morphologically and biochemically, is exceptional in that assembly of the virion appears to occur through a process involving de n o w formation of membranes in the cytoplasmic matrix (hummeler et al., 1967) . de novo formation of membranes is the usual process of assembly for members of the pox virus group (dales and mosbach, 1968) . the appearance of viral proteins on the cell surface, as well as the cellular site of virus assembly, can be modified by external agents including antibodies and lectins, the phenomenon of antigenic modulation involves altering the expression of cell surface antigens by specific antibody, and such antigens may be of viral origin (lampert et d . , 1975) . this undoubtedly has an effect on viral maturation, although the precise effects have not been determined. exposure of influenza virus-infected cells to convanavalin a appears to alter the site of virus maturation (stitz et al., 1977) . in the presence of this lectin, normal maturation at the plasma membrane is not observed, but instead large numbers of virions are observed in intracellular vacuoles. it is evident therefore that the maturation site for enveloped viruses can vary with the virus type as well as the host cell, and can be altered in response to specific stimuli. the process is likely to be determined as a result of interactions between virus-specific proteins and host cell membranes. i t is remarkable that in most instances only a single type of cellular membrane is selected as the site of virus assembly in a particular virus-infected cell. an understanding of the mechanisms which govern the selection of the assembly site may provide new insights into the assembly of cellular membrane components and organelles, since similar interactions are likely to be involved in determining the location of subsets of cellular proteins in specific cellular organelles. the synthesis and assembly of viral membrane components have been analyzed in cells in which host cell synthesis is inhibited as a result of virus infection. similar conclusions have been made for several virus types. viral glycoproteins appear to be synthesized on membrane-bound polyribosomes and remain associated with various cellular membranes (spear and roizman, 1970; compans, 1973a,b; stanley et al., 1973; klenket al., 1974; david, 1973; hay, 1974; atkinsonet al., 1976; nagai et al., 1976 ; knipe et al., 1977) . glycosylation occurs in association with cytoplasmic membranes. glycoproteins appear to migrate from rough endoplasmic reticulum to smooth or golgi complex membranes to the cell surface and are then incorporated into virions; at no time are they found as ''soluble'' cytoplasmic components. this general scheme for the synthesis and migration of proteins through intracellular membranes to the cell surface has been termed membrane flow. although the precise intracellular location of viral glycoproteins remains to be established, a scheme may be envisaged in which these components remain associated with the cisternal side of membranes of the endoplasmic reticulum and golgi complex after synthesis. incorporation into the plasma membrane by a process of vesicle fusion would then result in the correct orientation on the cell surface for viral as-sembly. a similar scheme has been suggested for the incorporation of glycoproteins into the plasma membrane, based on the distribution of oligosaccharides in different membrane fractions (hirano et al., 1972) . viral systems allowed investigators to follow the intracellular migration and glycosylation of specific glycoprotein species for the first time, whereas previous studies with cellular glycoproteins kad dealt with cell fractions essentially uncharacterized as to the specific proteins present. recently, katz et al. (1977) and rothman and lodish (1977) have described a new model system for studies of the incorporation of viral glycoproteins into membranes. katz et al. (1977) have shown that the mrna for vsv g-protein translated in vitro by wheat germ extracts in the presence of dog pancreas rough endoplasmic reticulum is associated with the membrane. furthermore, the newly synthesized g protein was shown to span the membrane with the amino-terminal asymmetrically located. in addition, the polypeptide portion which penetrated the membrane was shown to be glycosylated. glycosylation did not occur in the absence of membranes. these studies were extended by rothman and lodish (1977) , who showed that the insertion of g-protein into the membrane begins when 80 or fewer amino acid residues are polymerized. evidence was also obtained that the nascent chain is glycosylated while still attached to the ribosomes on the cytoplasmic side of the endoplasmic reticulum vesicle. since the mechanism by which viral glycoproteins are inserted into membranes is in all probability similar to that of the insertion of host cell glycoproteins, further studies using viral systems may provide more insight into the mechanism of insertion of glycoproteins into membranes. the process of glycosylation does not appear to play an important role in determining the intracellular migration of glycoproteins. although this has been suggested as a possible function for carbohydrate components of glycoproteins, the available data using inhibitors of glycosylation suggest that it is possible to inhibit or extensively modify the gl ycosylation process without preventing the migration of viral glycoproteins to the cell surface (courtney et al., 1973; nakamura and compans, 1978a) . the incorporation of carbohydrate-free m proteins into membranes appears to involve a distinct pathway in which cytoplasmic synthesis is followed by rapid association with the plasma membrane (lazarowitz et d., 1971; meier-ewert and compans, 1974; hay, 1974; nagai et al., 1976; knipe et al., 1977) . no evidence for migration through cytoplasmic membrane structures has been obtained' for these components, aad it has been suggested that they are inserted directly into membranes after synthesis. cytoplasmic synthesis followed by direct insertion into membranes has also been proposed for some classes of cellular membrane proteins (lodish and small, 1975) . although there are several unanswered questions concerning the precise steps in assembly even for the best studied enveloped viruses, the available information from electron microscope studies as well as the biochemical approaches described above suggest a scheme like that depicted in fig. 2 for influenza virus. glycoproteins appear to be inserted into the plasma membrane as the first step in assembly, after migration through cytoplasmic membranes. after they are inserted into the plasma membrane, initial random distribution may occur in which the proteins are free to undergo lateral diffusion in the plane of the membrane. such random distribution is illustrated for the glycoproteins of parainfluenza virus in fig. 3 , and similar observations have been reported for other viruses (birdwell and strauss, 1974; h. schwarz et al., 1976) . random distribution of viral antigens (fig. 3 ) is observed only when ferritin-antibody conjugates are applied to cells after glutaraldehyde fixation. in previous studies of the distribution of antigen, in which unfixed cells were used ), antigens were observed in discrete patches. it is likely that under these conditions they undergo lateral redistribution and aggregation into patches as a result of bivalent antibody. schematic diagram of the assembly process of an influenza virion. clycoproteins are thought to be inserted into the plasma membrane by a process called memhrane flow and are initially found randomly dispersed in the membrane. following in--sertion of the m protein, glycoproteins are thought to accumulate in discrete regions from which host cell membrane proteins are excluded. the association of the ribonucleoprotein (rnp) with such regions of modified membrane is followed by budding and release of the completed virion. in the case of influenza virus, the m protein may form a domain on the internal surface of the plasma membrane, stabilized by proteinprotein interactions. specific recognition of the m protein by the glycoproteins could then produce an accumulation of glycoproteins in a circumscribed region of the cell surface. alternatively, it is possible that m protein monomers associate with glycoprotein monomers at the plasma membrane, and that these complexes undergo lateral diffusion and aggregation into domains. in either case the lack of host cell membrane proteins in the viral envelope indicates that cellular proteins are efficiently excluded from the region of the plasma membrane which becomes the viral envelope. association of the nucleocapsid with regions of the cell surface containing viral envelope protein may stimulate the process of budding. fig. 4 . the presence of virus-specific glycoproteins on the external surface is indicated by tagging with ferritin-conjugated antibody. in fig. 5 , the emerging virus particles tagged with ferritin-antibody are shown at higher magnificafor viruses with icosahedral nucleocapsids and no m protein, a similar mechanism for virus assembly has been proposed in which the nucleocapsid itself binds to glycoproteincontaining membranes, with subsequent lateral diffusion and clustering of the glycoproteins in this region. although information is accumulating about the pathways by which viral membrane proteins are incorporated into plasma membranes, there is little direct evidence concerning the precise interactions which lead to the formation of domains on the cell surface which contain virus-specific proteins and lack host cell proteins. the lack of significant amounts of host cell protein in the virion indicates that such domains must he intermediates in assembly. further, the available data on viral protein composition (table 11 ) and morphology suggest that assembly interactions may differ for various virus groups. in all cases it is likely that protein-protein interaction serves to create a patch of virus-specific proteins in a cellular membrane, but this interaction may occur on the external or internal surface of the lipid bilayer. as discussed above, for viruses that contain m proteins or welldefined icosahedral nucleocapsids, some evidence has been obtained for the initial random distribution of glycoproteins on the cell surface, which may be followed by lateral diffusion and accumulation of glycoproteins in juxtaposition to the m protein or icosahedral nucleocapsid, with transmembrane interactions between the external and internal proteins serving to anchor the viral glycoproteins in place. with other virus types that contain a large amount of glycoprotein and no obvious m protein or well-defined icosahedral capsid, it is possible that lateral interactions between the glycoproteins are important in assembly. the glycoproteins of togaviruses and bunyaviruses have been observed in a regular surface arrangement (von bonsdorff and harrison, 1975; von bonsdorff and pettersson, 1975) . the latter viruses lack an m protein or an icosahedral internal component, and it has been suggested that direct interactions between glycoproteins may be involved in assembly and maintenance of the viral structure (von bonsdorff and pettersson, 1975) . similar interactions may be important in other virus groups in which glycoproteins are major protein constituents of the virion and there is no known internal membrane protein, such as b-type oncomaviruses and coronaviruses. pox viruses are unique in that formation of their lipid-containing membrane occurs de novo in the cytoplasm, rather than on a preexisting membrane structure. these viruses thus provide an unusual system for investigation of the molecular interactions involved in the formation of a highly organized structure within the cytoplasmic matrix (dales and mosbach, 1968 ). however, these viruses are structurally very complex, and limited information has been obtained on their molecular organization. shape and size determination may also be controlled by viral proteins at various levels; nucleocapsids, m proteins, or glycoproteins could be the determining factor for different virus groups. the phenomenon of phenotypic mixing of envelope glycoproteins in cells doubly infected with vsv and the parainfluenza virus sv5 clearly demonstrates that the internal proteins and not the glycoproteins determine the particle size and shape of rhabdoviruses mcsharry et al., 1971 ). this conclusion is supported by the observation that particles with the internal proteins of vsv possess the characteristic bullet shape of vsv while containing mixtures of envelope glycoproteins derived from vsv and sv5. a similar analysis of phenotypically mixed particles produced by dual infections with viruses of other major groups may provide further insights into the macromolecular interactions involved in virion assembly. the fixed shape and size of many lipid-containing viruses stands in contrast to the situation in membranous cellular organelles, which generally exhibit pleomorphism. similar pleomorphism is observed in some enveloped viruses. the assembly of such membranes of organelles and pleomorphic viruses may be regulated more by the production of materials than by precise constraints imposed by macromolecular interactions. morphology and entry of enveloped and deenveloped equine abortion (herpes) virus the amino acid and carbohydrate composition of the neuraminidase of b/lee/40 influenza virus assembly of vesicular stomatitis virus glycoprotein and matrix protein into hela cell plasma membranes morphogenesis of influenza a virus in ehrlich ascites tumor cells as revealed by thin-sectioning and freeze-etching enveloped viruses as model membrane systems: uicroviscosity of vesicular stomatitis virus and host cell membranes undisturbed release of influenza virus in the presence of univalent antineuraminidase antibodies differential action ofaspergillus glycosidase activities of influenza and newcastle disease viruses replication of sindbis virus. iv. electron microscopic study of the insertion of viral glycoproteins into the surface of infected cells dissociation of vesicular stomatitis virus and relation of the virion proteins to the viral transcriptase restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components theoretical aspects of structure and assembly of viral envelopes structural components of rna tumor viruses crystalline antigen from the influenza virus envelope biological parameters of fusion from without lipid and protein organization in vesicular stomatitis and sindbis viruses a,(n,) neuraminidase of the x-7 influenza virus recombinant: determination of molecular size and subunit composition of the active unit the biologically active proteins of influenza virus: neurarninidase. i n "the influenza viruses and influenza comparison of membrane protein glycopeptides of sindbis virus and vesicular stomatitis virus model for vesicular stomatitis virus phenotypic mixing of envelope proteins of the parainfluenza virus sv5 and vesicular stomatitis virus reproduction of paramyxoviruses. i n "comprehensive virology studies on proteolytic cleavage and glycosylation of the hemagglutinin of influenza a and b viruses. i n "negative strand viruses influenza virus proteins. 11. association with components of the cytoplasm distinct carbohydrate components of influenza virus glycoproteins in smooth and rough cytoplasmic membranes the structure and assembly of influenza and parainfluenza viruses reproduction of myxoviruses. i n "comprehensive virology incorporation of sulfate into influenza virus glycoproteins an electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus sv5. virolog!j 30 effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza a2 virus influenza virus proteins. i. analysis of polypeptides of the virion and identification of spike glycoproteins structure of the ribonucleoprotein of influenza virus assembly of lipid-containing viruses effects of 2-deoxy-dglucose on herpes simplex virus replication vaccinia as a model for membrane biogenesis antigenic characterization of two sindbis envelope glycoproteins separated by isoelectric focusing influenza virus assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane membrane receptors for murine leukemia viruses: characterization using the purified viral envelope glycoprotein, gp71 characteristics of two new avian tumor virus subgroups carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus grown in mammalian cell lines carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus partial structural analysis of the oligosaccharide moieties of the vesicular stomatitis virus glycoprotein by sequential chemical and enzymatic degradation epidemiological concepts and methods presence of murine leukemia virus envelope proteins gp70 and p15(e) in a common polyprotein of infected cells neutralization of homologous and heterologous oncornaviruses by antisera against the p 15(e) and gp7l polypeptides of friend murine leukemia virus the membrane proteins of semliki forest virus have a hydrophobic part attached to the viral membrane structural proteins of tacaribe and tamiami virions cross-linking of the spike glycoproteins in semliki forest virus with dimethyl suberimidate location of the spike glycoproteins in the semliki forest virus membrane isolation and characterization of the membrane proteins of semliki forest virus determining influence of helper virus on the host range and susceptibility to interference of rsv lipid and protein organization in sindbis virus the polypeptides of influenza virus. 11. interpretation ofpolyacrylamide gel electrophoresis patterns studies on the formation of the influenza virus envelope characteristics of sendai virus receptors in a model membrane model membranes and sendai virus: surface-surface interactions. i n "negative strand viruses proteins specified by herpes simplex virus. xii. the virion polypeptides of type 1 strains protein composition of coronavirus oc-43 distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: its implications for membrane biogenesis trypsin action on the growth of sendai virus in tissue culture cells. 111. structural difference of sendai viruses grown in eggs and tissue culture cells regulation of herpes virus macromolecular synthesis. i. cascade regulation of the synthesis of three groups of viral proteins structure and development of rabies virus in tissue culture glycosylation of vesicular stomatitis virus glycoprotein in virus-infected hela cells inhibition of avian sarcoma virus replication by glucosamine immunological relationships among envelope antigens of avian tumor viruses the oligosaccharide units of human type l immunoglobulin m (macroglobulin) interaction of sindbis virus glycoproteins during morphogenesis inhibition of the multiplication of enveloped rna-viruses by glucosamine and 2-deoxy-d-glucose synthesis of'proteins in cells infected with herpes virus. xi. sulfated structural proteins membrane assembly in vitro: synthesis, glycosylation, and asymmetric insertion of a transmembrane protein isolation of defective mutant of avian sarcoma virus sindbis virus glycoproteins: effect of the host cell on the oligosaccharides the effects of enzymes on structural and biological properties of semliki forest virus inhibition of influenza virus multiplication with a glucose antimetabolite (2-deoxy-d-glucose) viral envelopes and their relationship to cellular membranes lipids of plasma membranes of monkey and hamster kidney cells and of parainfluenza virions grown in these cells glycosphingolipids of plasma membranes of cultured cells and an enveloped virus (sv5) grown in these cells the proteins of the parainfluenza virus sv5 11. the carbohydrate content and glycoproteins of the virion an electron microscope study of the presence or absence of neuraminic acid in enveloped viruses on the structure of the influenza virus envelope inhibition of glycoprotein biosynthesis of influenza virus by d-glucosamine and 2-deoxy-~-glucose association of influenza virus proteins with cytoplasmic fractions activation of influenza a viruses by trypsin treatment a sedimentable component of allantoic fluid and.its relationship to influenza viruses separate pathways of maturation ofthe major structural proteins of vesicular stomatitis virus the interaction between virus and antibody. hi. examination of virus-antibody complexes with the electron microscope the polypeptides and rna of sendai virus antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy effect of membrane protein on the lipid bilayer structure: a spin label esr study of vesicular stomatitis virus spin label esr study of the lipid-containing membrane of influenza virus structure of the lipid phase of rauscher murine leukemia virus organization of the lipid phase in viral membranes: effects of independent variation of the lipid and the protein composition separation of two polypeptide chains from the hemagglutinin subunit of influenza virus amino acid composition of polypeptides from influenza virus particles morphology of the isolated hemagglutinin and neuraminidase subunits of influenza virus studies on the origin of pandemic influenza. 11. peptide maps of the light and heavy polypeptide chains from the hemagglutinin subunits of a, influenza viruses isolated before and after the appearance of hong kong influenza enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide influenza virus structural and nonstructural proteins in infected cells and their plasma membranes proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. function of the uncleaved polypeptide ha the polypeptide of influenza virus composition of the neuraminidase tunicamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis viruses the membrane structure of lipid-containing viruses organization of the membrane of standard and incomplete influenza virus membrane proteins synthesized by rabbit reticulocytes carbohydrate composition of vesicular stomatitis virus proteins ofvesicular stomatitis virus and of phenotypically mixed vesicular stomatitis virus-simian virus 5 virions transmembrane phospholipid motions induced by f glycoprotein in hemagglutinating virus of japan protein-bound oligosaccharides of semliki forest virus time course of synthesis and assembly of influenza virus proteins microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell vesicular stomatitis virus envelope glycoprotein a]-terations induced b y host cell transformation oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus glycoprotein fragment associated with vesicular stomatitis virus after proteolytic digestion bunyaviridae: morphologic and morphogenetic similarities of bunyamwera serologic supergroup viruses and several other arthropod-borne viruses activation of precursor to both glycoproteins of newcastle disease virus by proteolytic cleavage studies on the assembly of the envelope of newcastle disease virus proteolytic cleavage of the viral glycoproteins and its significance for the virulence of newcastle disease virus effects of inhibitors on glycosylation sulfation, and assembly of influenza virus glycoproteins glycopeptide components of influenza viral glycoproteins host cell dependent glycosylation of influenza virus glycoproteins primary structure and side chain interactions of pf, filamentous bacterial virus coat protein the structural proteins of la crosse virus molecular anatomy of herpes viruses: recent studies the fusion of ehrlich's tumor cells caused by hvj virus in uitro ultrastructure of animal viruses and bacteriophages inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-n-trifluoracetylneuraminic acid. (fana): mechanism of action isolation and characterization of influenza virus recombinants with high and low neuraminidase activity: use of 2-(3'-methoxy-pheny1)-n-acetylneuraminic acid to identify cloned populations characterization of temperature sensitive influenza virus mutants defective in neuraminidase sulfated glycoproteins and polysaccharides of enveloped viruses phospholipid composition of rous sarcoma virus, host cell membranes, and other enveloped viruses the replication of herpes viruses transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus role of carbohydrate in biological functions of friend murine leukemia virus gp7lj isolation and purification of the envelope proteins of newcastle disease virus identification of biological activity of paramyxovirus glycoproteins: activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus isolation of paramyxovirus glycoproteins: association of both hemagglutinating and neuraminidase activities with the larger sv5 glycoprotein identification of a second glycoprotein in sindbis virus grbwth of enveloped rna viruses in a line of chinese hamster ovary cells with deficientn-acetylglucosaminyl transferase activity sialoglycoprotein of vesicular stomatitis virus: role of the neuraminic acid in infecti0n mosquito cells infected with vsv yield unsialylated virions of low infectivity association of vesicular stomatitis virus glycoproteins with virion membrane: characterization of the lipophilic tail fragment detection of an unstable rna in chick fibroblasts after reduction of the utp pool by glucosamine the struchire of influenza virus. i. the polypeptides of the virion the biologically active proteins of influenza virus: the hemagglutinin properties of mouse leukemia virus. xi. immunoelectron microscopic studies on viral antigens on the cell surface inhibition of glycosylation of influenza virus hemagg1utinin carbohydrates of influenza virus i. glycopeptides derived from viral glycoproteins after labeling with radioactive sugars suppression of glycoprotein formation of semliki forest, influenza, and avian sarcoma virus by tunicamycin effect of the viral proteins on the fluidity of the membrane lipids in sindbis virus red cell membrane glycoprotein: amino acid sequence of an intramembranous region functional significance of sialidase during influenza virus multiplication isolation and characterization of two distinct types of hvj (sendai virus) spikes the fluid mosaic model of the structure of cell membranes studies on the primary structure of the influenza virus hemagglutinin. proc. n a t l the proteins specified by herpes simplex virus. iv. the site of glycosylation and accumulation of viral membrane proteins the polypeptides of influenza virus. vii. synthesis of the hemagglutinin studies on the inhibitory effect of lectins on myxovirus re1ease sc nuclear magnetic resonance studies on the lipid organization of enveloped virions (vesicular stomatitis virus) sialic acid content of sindbis virus from vertebrate and mosquito cells structural proteins of ribonucleic acid tumor viruses carbohydrate content of membrane protein of sindbis virus tunicamycin inhibition of polyisoprenyl n-acetylglucosaminyl pyrophosphate formation in calf liver microsomes strain-specific antigen of the avian leukosis sarcoma virus group asymmetry of influenza virus membrane bilayer demonstrated with phospholipase c studies on the amphipathic nature of the membrane proteins in semliki forest virus structural components of the arenavirus pichinde a heterogeneity of rous sarcoma virus revealed by selectively resistant chick embryo cells patterns of viral interference in the avian leukosis and sarcoma complex sindbis virus glycoproteins form a regular surface lattice surface structure of uukuniemi virus reproduction of rhabdoviruses estimation of the molecular weights of the polypeptide chains from the isolated hemagglutinin and neuraminidase subunits of influenza viruses studies on the origin of pandemic influenza. i. antigenic analysis of a2 influenza viruses isolated before and after the appearance of hong kong influenza using antisera to the isolated hemagglutinin subunits antigenic variation of influenza viruses influenza viral proteins: identification and synthesis evidence from studies with a cross-linking reagent that the haemagglutinin of influenza virus is a trimer structural components of mouse mammary vesicular stomatitis virus maturation og!/ 79,446-448. shape of influenza virus neuraminidase research by the authors was supported by grants no. a1 12680 and ca 18611 from the usphs, pcm76-09711 from the national science foundation, and vc 149b from the american cancer society. m.c.k. was supported b y a fellowship from the anna fuller fund. key: cord-008556-oetrdm8g authors: kozak, marilyn title: regulation of protein synthesis in virus-infected animal cells date: 2008-03-01 journal: adv virus res doi: 10.1016/s0065-3527(08)60265-1 sha: doc_id: 8556 cord_uid: oetrdm8g this chapter summarizes the structural features that govern the translation of viral mrnas: where the synthesis of a protein starts and ends, how many proteins can be produced from one mrna, and how efficiently. it focuses on the interplay between viral and cellular mrnas and the translational machinery. that interplay, together with the intrinsic structure of viral mrnas, determines the patterns of translation in infected cells. it also points out some possibilities for translational regulation that can only be glimpsed at present, but are likely to come into focus in the future. the mechanism of selecting the initiation site for protein synthesis appears to follow a single formula. the translational machinery displays a certain flexibility that is exploited more frequently by viral than by cellular mrnas. although some of the parameters that determine efficiency have been identified, how efficiently a given mrna will be translated cannot be predicted by summing the known parameters. the translation of viral mrnas: where the synthesis of a protein starts and ends, how many proteins can be produced from one mrna, and how efficiently. the next section focuses on the interplay between viral and cellular mrnas and the translational machinery. that interplay, together with the intrinsic structure of viral mrnas, determines the patterns of translation in infected cells. the final section points out some possibilities for translational regulation that can only be glimpsed at present, but are likely to come into focus in the future. to keep the project manageable, i have concentrated on animal viruses. plant viruses are mentioned, however, when they provide the best (or sometimes the unique) example of a given mechanism. the structural requirements for mrna function have been determined by inspection'of natural eukaryotic mrnas, followed by manipulation of features that looked suspicious. the general structural characteristics of eukaryotic mrnas have been reviewed previously (kozak, 1983a) and will not be elaborated here. the discovery of the m7g cap on a wide variety of viral and cellular mrnas (shatkin, 1976 ) was a provocative clue that the mechanism of initiation in eukaryotes differs from prokaryotes. although the list of plant virus mrnas that are translated without a cap has grown in recent years, picornaviruses and caliciviruses are still the only animal viruses known to be translated without a cap (nomoto et al., 1976; ehresmann and schaffer, 1979) . indeed, the near-indispensibility of the m7g cap may be inferred from the fact that animal viruses that replicate in the cytoplasm routinely encode their own capping and methylating enzymes. this is true not only for poxviruses (moss et d., 1976) , where the vast coding capacity of the genome allows room for frills, but also for reovirus (furuichi et al., 19761, vesicular stomatitis virus (vsv) (abraham et al., 19751, and alphaviruses (cross, 1983) ) in which the small size of the genome limits the encoded proteins to the barest essentials. the m7g cap enhances both the stability and translatability of mrnas. transcripts that are capped but not methylated are stable, but nonetheless untranslatable (furuichi et al., 1977; horikami et al., 1984) . much of the discussion that follows assumes that a scanning mechanism underlies the initiation process. the scanning model postulates that a 40 s ribosomal subunit binds initially at the 5' end of the mrna viral translation 23 1 and migrates until it reaches the first aug triplet. if the first aug codon occurs in the optimal context (accaugg-see kozak, 1981a kozak, , 1984a kozak, , 1986a all 40 s subunits stop there, and that aug serves as the unique site of initiation. if the first aug triplet occurs in a suboptimal context, only some 40 s subunits will initiate there; some will migrate beyond that site and initiate at an aug codon farther downstream. the scanning hypothesis is not universally accepted, but it is supported by extensive evidence from many laboratories (reviewed by kozak, 1980 kozak, , 1981b kozak, , 1986a . two alternative models have been suggested from time to time. one is that ribosomes bind directly to the sequence around the aug codon, but experiments designed to distinguish between scanning and direct binding do not support the latter (kozak, 1979a (kozak, , 1983b . a hybrid mechanism in which 98% of the ribosomes scan from the 5' end, while 2% of the binding occurs directly at the aug start site, is difficult to rule out, however. another suggestion is that secondary structure might guide the choice of aug codons (this idea is evaluated a few paragraphs hence). one consequence of the scanning mechanism is that deleting the "ribosome binding site" (i.e., the normal initiator codon and flanking sequences) will not abolish translation; ribosomes will simply use the next aug codon downstream, which, in some cases, has been shown to direct the synthesis of a biologically active, truncated protein (downey et al., 1984; halpern and smiley, 1984; katinka and yaniv, 1982) . conversely, introducing spurious upstream aug codons will reduce initiation from the authentic start site-a prediction that has been verified many times with laboratory constructs (bandyopadhyay and temin, 1984; lomedico and mcandrew, 1982; smith et al., 1983; zitomer et al., 1984) as well as with naturally occurring variant forms of mrna from the early1 and late regions of simian virus 40 (sv40) (barkan and mertz, 1984) . when the context around an upstream aug codon conforms closely to the accaugg consensus sequence, initiation from the downstream site is suppressed almost completely (kozak, 198313, 1984b; liu et al., 1984; m. scott and h. varmus, personal communication) . when the context around the upstream aug codon is less ideal, initiation from the downstream site is reduced but not abolished (kozak, 1986a) . stated in a more positive way, when the 5'-proximal aug codon occurs in a suboptimal context, ribosomes are able to initiate at the first and the second aug codons. this "leaky" scanning process is further explained and documented in section i1,c. the scanning mechanism predicts that translation should be downregulated by any ploy that interferes with the linear movement of 40 s ribosomal subunits from the cap to the aug codon: binding of a protein to the 5'-noncoding sequence; introducing spurious out-of-frame aug codons, as mentioned above; annealing cdna fragments that are complementary to the 5'-untranslated sequence (haarr et al., 1985; perdue et al., 1982; privalsky and bishop, 1982; willis et al., 1984) ; or creating a stable hairpin anywhere upstream from the aug codon, as described in the next section. on the other hand, the simplicity of the scanning mechanism suggests few possibilities for enhancing translation. although we know what features should be absent from the leader for a message to be efficient, the only features known to contribute in a positive way are the m7g cap and the sequence directly flanking the initiator codon. a promising place to look for other positive effectors is the tripartite leader on late adenovirus mrnas. transposition of the 200-nucleotide tripartite leader sequence to heterologous mrnas stimulates their translation 20-fold (berkner and sharp, 1985; logan and shenk, 1984) , but the feature responsible for the stimulation has not been pinpointed, and could turn out disappointingly to be a long sequence that simply lacks all of the negative effectors cited above. the impression that the leader sequences on most viral mrnas do not contain unidentified translational "enhancers" is reinforced by the ease with which 5'-noncoding sequences can be deleted without deleterious effects (bendig et al., 1980; spindler and berk, 1984a; villarreal et al., 1979) .2 if our intuition is correct that "extra" 5'aoncoding sequences are more likely to inhibit than to help, the trend toward short 5'-noncoding sequences on many viral mrnas becomes significant (reviewed by kozak, 1981b ; see also rose1 and moss, 1985) . indeed, the 24-nucleotide leader sequence on the mrna that encodes adenovirus polypeptide ix seems to mediate translation more efficiently than the long tripartite leader that has received so much attention (lawrence and jackson, 1982) . the synthesis of polyoma virus t antigen was significantly reduced in only one of the mutants studied by bendig et al. (1980) -a mutant in which the deletion extended to within two nucleotides of the aug codon. this fits with evidence from other sources that (only) the nucleotides immediately preceding the aug codon are part of the ribosome recognition sequence. secondary structure in viral mrnas might have various effects on translation. 1. one might expect secondary structure to inhibit more when it occurs near the cap, which is the presumptive entry site for ribosomes, than when a hairpin occurs farther downstream, because 40 s ribosomal subunits once bound must be able to melt secondary structure to some extent. (one knows for sure that 80 s ribosomes melt secondary structure during the elongation phase of protein synthesis; the triplet code could not be read linearly otherwise.) the prediction that 40 s ribosomal subunits can melt their way through secondary structure within the interior of the leader sequence has been verified: introducing a 13-base-pair hairpin (ag -30 kcal/mol) 60 nucleotides downstream from the cap did not impair the translation of preproinsulin mrna in uzuo (kozak, 1986b) . the effects of secondary structure close to the cap have not yet been tested systematically, but it has been noted that the 5' end of alfalfa mosaic virus rna-4 is unfolded (gehrke et al., 1983) and rna-4 is a notoriously efficient message. godefroy-colburn et al., (1985b) claim more generally that the degree of cap accessibility of the four alfalfa mosaic virus mrnas correlates with their translational efficiency, but the correlation appears weak. the cap was indeed least accessible on rna-3, which ranks lowest in translational efficiency, but the cap was equally accessible on rnas 1,2, and 4, which differ 15fold in competitive efficiency (godefroy-colburn, 1985a) . 2. although we expect ribosomes to melt secondary structure to some extent, there must be a limit to that ability. whereas a hairpin of -30 kcalimol at the midpoint of the leader sequence (involving neither the cap nor the aug codon) did not reduce the synthesis of preproinsulin under normal culture conditions, a hairpin of -50 kcal/mol nearly abolished translation (kozak, 198613) . because the hairpin did not encroach on the aug codon, the observed inhibition seems incompatible with the direct-binding hypothesis, but is consistent with the scanning hypothesis. pelletier and sonenberg (1985) have also shown that translational efficiency decreases as secondary structure in the 5'noncoding region increases. 3. there is no experimental support for the idea that secondary structure orients the cap and the aug codon, thus determining which aug will initiate translation. were that true, denaturation should impair translation; in fact, denaturation often enhances (payvar and schimke, 1979) . nor is there support for the idea that downstream cistrons are silent due to conformational constraints: attempts to activate internal initiation sites by denaturing viral mrnas invariably fail (collins et al., 1982; monckton and westaway, 1982) . a popular idea is that when secondary structure sequesters the 5'-proximal aug triplet, it might be skipped by ribosomes in favor of the next exposed aug codon (darlix et al., 1982; ghosh et al., 1978; hay and aloni, 1985; nomoto et al., 1982) . the results of a direct test contradict that notion, however, when the primary sequence around the 5'-proximal aug codon in a chimeric preproinsulin mrna was favorable for initiation, no translation from a downstream site could be detected irrespective of whether the first aug codon was single stranded or base paired (kozak, 1986b) . thus, 40 s ribosomal subunits appear to scan linearly, melting the secondary structure (ag 5 -30 kcal/mol) t o reach each aug codon in turn. if a hairpin is too stable to be melted (ag 2 -50 kcal/mol), the 40 s subunit apparently stalls, but it does not "jump over" the barrier. 4. in some viral mrnas, sequences at the 3' end are complementary, to a limited extent, to those at the 5' end (antczak et al., 1982; dasgupta et az., 1980) . that arrangement might be expected to inhibit translation-an expectation that has been confirmed recently using mrnas with artificially constructed terminal complementary sequences (spena et al., 1985) . some viruses seem to take measures to preclude such inhibition. whereas the genomic rnas of influenza (robertson, 1979) and bunyaviruses (eshita and bishop, 1984) have complementary 5'and 3'4erminal sequences, that potentially deleterious structure is not copied into mrna, inasmuch as the 3' terminus of each mrna stops short of the 5' end of the template strand (bouloy et al., 1984; eshita et al., 1985; hay et al., 1977) . arenaviruses also produce mrnas that lack the complementary sequences present at the termini of genomic rna (auperin et al., 1984) . 5 . incubation in hypertonic culture medium has been used often to study protein synthesis in virus-infected cells (see yates and nuss, 1982, and references therein) . hypertonic shock results in the rapid and reversible inhibition of protein synthesis at the level of initiation (saborio et al., 1974) . an intermediate concentration of salt or sucrose permits a residual low level of translation, under which circumstance viral protein synthesis nearly always predominates over cellular protein synthesis (cherney and wilhelm, 1979; nuss et al., 1975; oppermann and koch, 1976) . it is difficult to deduce the mechanism of this differential response from inspection of natural forms of viral and cellular mrnas. however, a cloned preproinsulin gene has been experimentally converted from hypertonic resistant to hypertonic sensitive by inserting into the 5'-noncoding sequence the oligonucleotide agcttgggccgtggtgg, thereby creating a 13base-pair hairpin around the aug initiator codon (mutant b13hp in kozak, 1986b) . a reasonable interpretation is that the hairpin structure (ag -30 kcal/mol), which does not inhibit translation under nor-ma1 culture conditions, is stabilized under hypertonic conditions to the point where it becomes inhibitory. an alternative explanation, currently under investigation, is that the primary sequence of the oligonucleotide insert underlies the enhanced sensitivity of mutant b13hp to hypertonic stress. if the first explanation turns out to be correct, one might suggest by extrapolation that most viral mrnas are less structured near the 5' end than are most cellular mrnas, and for that reason viral mrnas are more resistant to hypertonic stress. herpes simplex virus mrnas are a notable exception: they are unusually sensitive to hypertonic inhibition (stevely and mcgrath, 19781 , perhaps because their high g + c content generates extensive secondary structure. 6. the mechanism of action of interferon is too complex to discuss here, except to mention that double-stranded regions of rna, either free or incorporated into the mrna structure (debenedetti and baglioni, 1984; knight et al., 19851 , are critical in activating and targeting the interferon-induced enzymes. the deleterious effects of interferon on the stability and translation of viral mrnas have been reviewed by lengyel (1982) . the monocistronic rule means more than simply producing one protein from one mrna. a number of viral mrnas encode two or more proteins in nonoverlapping reading frames; with few exceptions, however, (see section ii,c), it is exclusively the 5'-proximal cistron that gets translated (shih and kaesberg, 1973; reviewed by kozak, 1978; . to cope with the usual inability of eukaryotic ribosomes to initiate at internal sites in mrna, the genomes of animal viruses are punctuated at one of four levels, as described below. the structures of plant virus rna genomes and their patterns of expression have been reviewed by davies and hull (19821 , and they are not exceptional. the mode of expression of cauliflower mosaic virus, which has a circular dna genome, is exceptional indeed, and is discussed in section i1,c. the following descriptions are generalized; additional details and references have been published elsewhere (kozak, 1981b) . each virus is classified according to its major mode of punctuation, which is often not the exclusive mode. 1. the genome itself is segmented. each segment typically consists of one gene, which is transcribed end to end, or nearly so. there is usually a simple correspondence between the size of the mrna and the size of the mature protein derived therefrom. reoviruses, influenza viruses, and bunyaviruses fit this description. arenaviruses and nodaviruses (e.g., black beetle virus) have segmented rna genomes but rely also on other mechanisms. 2. the viral genes are linked, but internal start and stop sites for transcription generate a separate mrna for each protein. punctuation is accomplished for the most part at the level of transcription rather than by posttranscriptional processing. again, the size of the mrna usually corresponds to the size of the mature p r~t e i n .~ this group includes poxviruses, herpesviruses, rhabdoviruses (vsv), and paramyxoviruses. here the genome lacks internal transcriptional and translational stoplstart sites. the genome-sized mrna is translated end to end to produce a "polyprotein," more than 2000 amino acids in length, which is cleaved to generate the mature viral proteins. the extreme situation in which all viral proteins are derived from a single precursor is characteristic of picornaviruses and flaviviruses (castle et al., 1986 ; c . m. . [rice et al. (1986) present a lucid explanation of some older data that had suggested a different translational strategy for flaviviruses.] posttranslational cleavage supplements other modes of punctuation in many animal virus systems, and is especially important in the maturation of retrovirus and alphavirus proteins. 4. the fourth, rather heterogeneous group of viruses characteristically produce big transcripts that cannot be translated completely: ribosomes bind at the 5' end and translate only up to the first stop codon, and the downstream cistrons in these polycistronic mrnas are usually silent. the downstream cistrons become translatable when they are moved closer to the 5' end, which is accomplished by producing truncated or subgenomic mrnas. various mechanisms generate these shortened transcripts. conventional splicing of nuclear transcripts is used by retroviruses, papovaviruses, and parvoviruses. adenoviruses also use splicing, on a rather grand scale (nevins, 1982; ziff, 1985) . coronaviruses use a novel cytoplasmic fusion mechanism to transfer a common leader sequence to each of six, progressively shorter, subgenomic mrnas (budzilowicz et al., 1985; lai et al., 1984; spaan et al., 1983) . in the case of alphaviruses and parvoviruses, initiation at a n 3 whereas the molecular weight correlation between mrnas and proteins holds for most early vaccinia virus genes (cooper and moss, 1979; hruby and ball, 1982) , late vaccinia mrnas are notoriously heterogeneous in size, apparently because transcription does not terminate discretely (mahr and roberts, 1984; rose1 and moss, 1985) . the 3'-proximal portions of such transcripts are assumed to be translationally silent. in the case of herpes simplex virus, the size of many mrnas corresponds simply to the size of the encoded protein, but more complex mrnas also exist (wagner, 1985) ; the functional significance of the latter is not yet clear. internal transcriptional promoter produces the subgenomic mrnas that encode the major capsid proteins (brzeski and kennedy, 1978; janik et al., 1984) . hepadnaviruses (hepatitis b and others) cannot yet be classified, since mrnas have been identified for some but not all of the viral proteins (tiollais et al., 1985) . the major subgenomic mrna is initiated at an internal promoter, and there is no evidence for splicing. the heterogeneous initiation sites for transcription in hepatitis viruses might be a means to regulate translation, as suggested by laub et al. (1983) and enders et al. (1985) . arenaviruses are a special case. the genomic s-rna segment codes for two structural proteins, n and gpc, but only gpc can be translated conceptually directly from the 5' half of virion rna; the 3' half of the sequence is an antisense version of the n gene (auperin et al., 1984) . thus, a subgenomic complementary mrna is produced to translate the n protein. although gpc could in theory be translated from the full-length viral s-rna, a subgenomic rna corresponding to the 5' portion of s-rna is also present in infected cells. this might be necessary to avoid "hybrid arrest" which could occur if translation were attempted with full-length viral and antiviral transcripts. whereas most eukaryotic mrnas are functionally monocistronic, certain viral mrnas have been shown to synthesize two separately initiated polypeptides. with few exceptions4 we can rationalize the 4 the mechanisms outlined herein cannot explain the (inefficient) internal initiation that occurs in a mutant form of rous sarcoma virus src mrna (mardon and varmus, 1983) . poliovirus mrna also initiates translation at more than one site, at least in uitro (celma and ehrenfeld, 1975) , but one cannot attempt an explanation until the sites have been identified. [dorner et al. (1984) claim to have localized an internal initiation site, but they did not prove that the template rna was intact. the fact that they could demonstrate "internal initiation" in extracts from reticulocytes but not from poliovirusinfected cells hints of an artifact.] because the poliovirus 5'-noncoding sequence has eight aug triplets upstream from the major translational start site (kitamura et al., 1981; racaniello and baltimore, 1981) , spurious initiation events are expected in that region. on the other hand, the upstream aug triplets would not preclude initiation of the polyprotein from the ninth aug codon, because seven of the upstream aug triplets lie in a weak context; the only one that lies in a favorable context is followed by an inframe terminator codon, which would allow reinitiation. the same explanations are compatible with the genomic sequences of many other picornaviruses callahan et al., 1985; forss et al., 1984; linemeyer et al., 1985) . the two structural peculiarities of picornavirus mrnas-presence of upstream aug codons and absence of production of two proteins from a single mrna by invoking one of the following mechanisms, each of which is experimentally supported. these mechanisms (with the exception of reinitiation) might be considered errors, i.e., the results of imprecise execution of some step in translation. a system that functions with less-than-perfect fidelity apparently gains the advantage of versatility. the scanning model postulates that, when the 5'-proximal aug codon occurs in a suboptimal context, ribosomes will initiate at that site as well as at another aug codon farther downstream. several nucleotides near the aug codon are known to affect the efficiency of initiation, but the most important determinants are a purine (preferably a) in position -3, and g in position +4; we can predict the occurrence of leaky scanning by focusing on those two positions. in each of the bifunctional viral mrnas listed in fig. 1 , the more 5'-proximal initiation site lies in a suboptimal context, thus rationalizing the ability of some ribosomes to reach the start of the second cistron. (in sv40 16 s mrna, influenza b, and adenovirus-12, which are bracketed in the center of the figure, the sequence flanking the first aug codon is not really weak, but it is not perfect; thus, some 10-20% of the 40 s subunits are expected to bypass the first aug codon and reach the second. that may be adequate to produce the second protein in the case of adenovirus and influenza virus, but it does not seem adequate to explain the synthesis of sv40 vp1, which is an abundant protein. in sv40 16 s mrna, however, ribosomes can reinitiate at the vp1 start site, as explained below.) the scanning model does not necessitate that the second aug codon lie in a stronger context than the first, although that usually is the case; it is necessary only that the first aug codon lie in a context that is less than optimal. each mrna listed in the upper part of fig. 1 produces two unrelated proteins, translated from two different reading frames. the mrnas in the lower part of the figure initiate at two aug codons in the same reading frame, thereby producing long and short versions of the same protein. whereas the relaxed scanning mechanism accounts qualitatively for the dual function of the mrnas listed in fig. 1 , the model is not very a cap-might be related it is possible that, when cap binding proteink) are not part of the 40 s initiation complex, aug codons in suboptimal contexts are recognized even less efficiently than usual, and the barrier effect of the upstream aug codons in poliovirus mrna would thus be minimized. perhaps p220 is cleaved (see section iii,d) to directly facilitate viral translation, rather than to inhibit host translation. good a t predicting the frequency with which ribosomes initiate a t each site. one problem is that the ratio of initiation a t sites 1 and 2 in vivo is often different from that i n vitro (bos et al., 1981; clarke et al., 1985; dethlefsen and kolakofsky, 1983; , and the ratio changes when salt or other reaction conditions are varied. that is hardly surprising because the fidelity of initiation in vitro is sensitive to reaction conditions (jense et al., 1978; kozak, 1979b; petersen and hackett, 1985) . on the other hand, the in vivo ratio might be skewed if one protein is less stable or less efficiently extracted than the other. in addition to the obvious economy of using one mrna to make two proteins, in a fixed ratio, their simultaneous production might allow the polypeptides to interact as the nascent chains grow. it would be amusing to determine whether complementation is less efficient when two proteins that are normally translated from one mrna are instead synthesized from separate templates. the hundreds of eukaryotic cellular genes that have been sequenced to date invariably initiate translation a t aug. when alternate initiator codons were tested experimentally, however, they were not inert. eukaryotic ribosomes can initiate a t gug (kozak, unpublished data) and uug (zitomer et al., 1984) , but the efficiency is a t least 30-fold lower than at an aug codon in the same context; initiation at gug, uug, or other nonstandard codons is (barely) detectable only when the codon is preceded by the optimal a in position -3 (m. k., unpublished data). there is credible, albeit not definitive, evidence that alternate initiator codons are used in two virus systems to produce minor virion components. one is adeno-associated virus capsid protein b, which probably initiates a t an acg codon that lies upstream from the major aug start site (becerra et al., 1985) . [an acg codon in coliphage t7 mrna is also recognized as an initiator codon by wheat germ ribosomes i n vitro (anderson and buzash-pollert, 1985) . although the template is unnatural in that case, the evidence for initiation a t acg is irrefutable.] the second natural example is gpr80gag, a nonessential but nonetheless conserved form of gag produced by moloney murine leukemia virus (edwards and fan, 1980; the nucleotide sequence of the region is given by shinnick et al., 1981) . gpr80gag is analogous to the elongated form of gag produced by feline leukemia virus, except that the latter is presumably initiated at an upstream aug codon in a weak context (fig. l ) , whereas in murine leukemia virus the most likely initiation site(s) are upstream gug and/or cug codons that lie in a favorable context. charles van beveren has shown that gpr80gag is produced not only by the left-most column shows that in most cases the sequence around the first functional initiator codon is suboptimal with respect to the nucleotides in positions -3 and +4, thus explaining how some 40 s ribosomal subunits can reach the second initiation site. in the case of the coronaviruses and black beetle virus, the indicated proteins are predicted but have not yet been demonstrated. although the 6.7-kda protein predicted from adenovirus region e3 has not been seen, its ribosome binding site has been proven functional by demonstrating the synthesis of a fusion protein from an appropriately engineered mutant virus (wold et al., 1986) . all of the other proteins listed here have been detected in infected cells, and most have also been synthesized in cell-free translation systems. notes: %since the 5' ends of hepatitis virus and some bunyavirus mrnas are heterogeneous (laub et al., 1983; patterson et al., 19831 , the second protein could be translated, without invoking leaky scanning, from the portion of the mrna population that lacks upstream aug codons. binfluenza virus rna-6 is unusual in that the first and second aug codons are separated by only four nucleotides (shaw et al., 1982) , but that probably does not explain the ability of ribosomes to initiate at both sites. in a version of preproinsulin mrna in which the first and second aug codons (both in the perfect context for initiation) were moloney virus, but also by two other murine leukemia viruses that have no aug codons upstream from the major (pr65gag) start site (personal communication). thus, there is no alternative to believing that nonstandard codon(s) are used to initiate the elongated form ofgag. experiments to pinpoint the start sites are in progress in van beveren's laboratory. although reinitiation was documented years ago in prokaryotes, there was no reason to suspect a similar phenomenon in eukaryotes until laboratory manipulations with cloned genes yielded some results separated by five nucleotides, ribosomes were unable to initiate a t the second member of the pair (kozak, 1984b) . 'because the first reading frame terminates upstream from the second in sv40 mrnas, ribosomes could reach the start site for vp1 by a combination of leaky scanning and reinitiation. dthe arrangement of aug codons near the 5' end of sv40 late 19 s mrna is gccaugg (out-of-frame at position 253-255) . . . uccaugg (start of vp2) . . . ccuaugc (out-of-frame a t position 679-681) . . . ggaaugg (start of vp3) . we postulate that leaky scanning allows some 40 s ribosomal subunits to bypass the first aug triplet (position 253-255) in order to initiate vp2. that does not contradict the fact that, in 16 s mrna, the aug codon in position 253-255 initiates the agnogene product. by extrapolating from the systematic measurements carried out in another system (kozak, 1986a) , we would expect 80-9070 of the ribosomes to initiate a t the aug codon in position 253-255, while 10-209 should reach the next aug; that seems sufficient to produce vp2, which is a minor component of the virion. synthesis of vp3 might depend on leaky scanning (bypassing the first three aug codons) as well as reinitiation, inasmuch as ribosomes that initiate a t the first aug codon would terminate before reaching the vp3 start site. ethe nucleotide in position -3 varies among strains of foot-and-mouth disease virus, and the relative yields of p20a and p16 vary accordingly (clarke et al., 1985) . fit is likely that two annaug sequences farther upstream are also used to produce longer forms of surface antigen (heermann et al., 1984) . most transcripts lack the extreme upstream aug codons, however. nthe two proteins postulated for feline leukemia virus are indeed seen in infected cells, but the mechanism of synthesis postulated here has not been proven. infrequent initiation at weak, upstream aug codons is also suspected with mrnas from some other retroviruses (gruss et al., 1981; willumsen et al., 1984) . references: sendai virus: giorgi et al., 1983 . measles virus: bellini et al., 1985 . reovirus: cashdollar et al., 1985 ernst and shatkin, 1985; kozak, 1982; sarkar et al., 1985. bunyaviruses: eshita and fuller et al., 1983 pasek et al., 1979; persing et al., 1985 . feline leukemia virus: laprevotte et al., 1984 . herpes simplex: haarr et al., 1985 wagner et al., 1981. that are difficult to explain ~t h e r w i s e .~ the principal observation is that eukaryotic ribosomes can initiate at an internal aug codon, when another aug codon occurs upstream and in a highly favorable context (thus ruling out leaky scanning), provided that a terminator codon occurs in-frame with the first aug codon and upstream from the second (kozak, 1984b; liu et al., 1984; m. scott and h. varmus, personal communication) . we envision that when a complete "minicistron," i.e., an aug triplet followed by a terminator codon, occurs upstream, it is translated; but the 80 s ribosome does not detach at the terminator codon. rather, the 60 s subunit dissociates while the 40 s subunit remains bound to the message and resumes scanning. when the 40 s subunit reaches the next aug codon, it reinitiates translation. reinitiation is more eficient when the terminator codon precedes, rather than when it overlaps, the aug codon (m. kozak, unpublished) . with respect t o natural mrnas rather than laboratory constructs, elegant genetic manipulations implicate reinitiation in the translation of rous sarcoma virus src mrna (hughes et al., 1984) and cauliflower mosaic virus mrna , dixon et al., 1986 . the latter is the most striking example to date of a functionally polycistronic mrna in eukaryotes. the overlapping arrangement of cistrons rules out the possibility of reinitiation with many other viral mrnas (contreras et al., 1977; meshi et al., 1983; schwartz et al., 1983; . however, in some instances in which adjacent cistrons do not overlap, and reinitiation is therefore expected, it has not been observed (barker et al., 1983; goelet et al., 1982; knowland, 1974; ou et al., 1982) . reinitiation, together with leaky scanning, could theoretically account for translation of the sv40 agnogene pro-5 the alternative to reinitiation is to postulate that eukaryotic ribosomes can initiate directly at an internal aug codon, and that they usually fail to do so only because the downstream site is occluded by the stream of 80 s ribosomes advancing from upstream. occlusion indeed occurs during the translation of polycistronic prokaryotic transcripts, but the inhibitory effect of an overlapping upstream cistron is sometimes only twoor threefold (das and yanofsky, 1984; hoess et al., 1980) . berkhaut et al. (1985) claimed to see complete inhibition of translation of the ms2 lysis protein when the coat protein cistron overlapped, but the unknown sensitivity of their biological assay complicates the interpretation. moreover, their claim that a strong upstream initiation site (for coat protein) suppresses initiation from the much weaker site for lysis protein hardly compares with the situation in eukaryotes, where an upstream aug codon can completely suppress initiation from an equally favorable downstream site (kozak, 1983b, 198413) . the essential difference between the occlusion and reinitiation mechanisms is that the former postulates direct binding of ribosomes to internal aug codons, while the latter prohibits such binding. there is experimental evidence against direct binding (kozak, 1979a (kozak, , 1983b and against occlusion (kozak, 198413) . tein and vp1 from the same mrna, although neither mechanism has been experimentally demonstrated with sv40. (the simultaneous occurrence of two phenomena complicates the task of demonstrating either one.) reinitiation is expected within the leader region of rous sarcoma virus genomic rna, where three small open reading frames (orfs), one of them headed by an aug codon in a highly favorable context, precede the gag coding sequence (schwartz et al., 1983) . it has been difficult to demonstrate synthesis of the predicted leader peptides, perhaps because their small size makes them unstable. with admirable persistence, however, hackett et al. (1986) have devised a sensitive assay with which they have detected small amounts of the peptide encoded in the first minicistron of rous sarcoma virus. parenthetically, when one is designing experiments to probe the function of a particular viral or cellular product, one must remember that introducing a nonsense codon near the beginning of a gene might not abolish its function. if an in-frame aug codon occurs downstream from the nonsense codon, ribosomes will probably reinitiate and the truncated polypeptide might be functional. the mechanism by which reverse transcriptase is synthesized has long puzzled retrovirologists. the pol coding sequence is not preceded by an initiator codon; rather, reverse transcriptase is derived by cleavage from a joint gag-pol precursor (murphy et al., 1978; oppermann et al., 1977) . the problem is that the genomic arrangement of gag and pol sequences would seem to preclude their joint translation. in avian retroviruses, gag and pol are in different, partially overlapping, reading frames (schwartz et al., 1983) ; in murine retroviruses, gag and pol are in the same frame but are separated by a terminator codon (shinnick et al., 1981) . in both cases, the solution involves a translational "error." with avian retroviruses, about 5% of the ribosomes shift reading frames somewhere near the end of the gag sequence, thereby producing from one message both gag and a small amount of the gag-pol fusion protein. jacks and varmus (1985) have shown beyond reasonable doubt that frameshifting occurs near the gag-pol junction in a cell-free translation system from reticulocytes. by using mrna that was transcribed in uitro from cloned rous sarcoma virus dna, they excluded the possibility that a low-abundance, spliced transcript served as the template for the fusion protein. inspection of the gag-pol junction sequences in several other retroviruses leads one to expect that frameshifting is not limited to the avian system. neither is it limited to eukaryotes, of course. frameshifting occurs under intrigu-ing circumstances in a few bacterial and phage genes (craigen et al., 1985; dunn and studier, 1983; kastelein et al., 1982) . the excitement that accompanied the old discovery of a "readthrough" version of coliphage qp coat protein (weiner and weber, 1973) has been rekindled recently by finding a similar phenomenon in eukaryotic systems. in murine retroviruses, for example, the gag and pol sequences are separated by a single uag terminator codon, the occasional suppression of which generates a gag-pol fusion protein. the first hint of this came from supplementing a cell-free translation system with yeast suppressor trna, which indeed enhanced the synthesis of the gag-pol precursor (philipson et al., 1978) . the notion was confirmed for both murine and feline leukemia viruses when yoshinaka et al. (1985a,b) directly determined the amino acid sequence of the protease that constitutes the nh,-terminal portion of the pol gene product. suppression of a terminator codon is not peculiar to retroviruses, for it occurs also with alphaviruses (lopez et al., 1985; strauss et al., 1983) , tobacco mosaic virus (pelham, 1978) , and probably carnation mottle virus (guilley et al., 1985) . suppression of the uag codon in tobacco mosaic virus rna has been traced to the major tyrosine-specific trnas which, in tobacco cells, have the anticodon sequence gjia (beier et al., 1984a,b) . the most abundant trnaer from wheat germ has the highly modified queuine base (q) in place of g in the wobble position of the anticodon, and it is not able to suppress. thus, minor differences in trna structure can be an important determinant of host range for some viruses. in one sense, suppression solves the problem of how to produce a full-length protein from an interrupted coding sequence. but that probably misplaces the emphasis. the real problem might be how to produce only a small amount of an essential protein that might be toxic if overproduced. an inefficient mechanism, such as suppression or frameshifting, is an ideal solution. whereas the features described in the preceding section are intrinsic to viral mrnas, and can be demonstrated readily in a "universal" reticulocyte lysate, the translation of viral mrnas in uiuo is influenced by specific conditions that prevail in the cytoplasm of infected cells. the way in which the translational machinery is partitioned viral translation 245 between viral and host mrnas is one important consideration. because the literature concerning inhibition of host protein synthesis by animal viruses has already been reviewed at length (fraenkel-conrat and wagner, 1984; kaariainen and ranki, 1984; shatkin, 1983) , i shall be selective in my coverage. an overview of the phenomenology is presented in table i . the general mechanisms of host shutoff defined by these phenomena are described briefly in sections b and c, which are followed by a detailed discussion of two viruses-poliovirus and adenovirus-that seem to merit more attention. the phenomenon of host shutoff is not as widespread as might appear from table i . retroviruses, paramyxoviruses, parvoviruses, and flaviviruses do not suppress host translation, and papovaviruses actually stimulate host protein synthesis. because host shutoff is interesting, and because it is easier to detect viral protein synthesis against a clean background, virologists understandably have focused on systems that demonstrate the phenomenon. the inhibition of host protein synthesis may be of more interest to virologists than to viruses, however. in many cases, the yield of infectious progeny from a virus that fails to shut off host protein synthesis is the same as from another virus strain (or the same virus in a different cell line) in which host protein synthesis is obliterated (detjen et al., 1982; gillies and stollar, 1982; jen and thach, 1982; lodish and porter, 1981; minor et at., 1979; munemitsu and samuel, 1984; read and frenkel, 1983; sharpe and fields, 1982) . a virus strain that suppresses host macromolecular synthesis sometimes replicates faster in culture than one that does not, however. whether the inhibition of host protein synthesis is beneficial or harmful or irrelevant to the virus during the course of natural infections is not known. in short, with a few viruses inhibition of host protein synthesis might be a strategic move, necessary for efficient expression of viral genes, but no unequivocal example can be cited. in most instances, host shutoff is likely to be an unintentional side effect of viral gene expression-an effect of no real value, and possibly even harmful, to the virus. it is interesting that poliovirus replicates better during coinfection with cytomegalovirus than during single infection; cytomegalovirus stimulates the cell functions that are turned off by poliovirus (furukawa et al., 1978) ! there are examples of nonpermissive virus-cell systems in which macromolecular synthesis is inhibited so effectively that neither host nor viral proteins can be made (brown and moyer, 1983; drillien et al., 1978; jones et al., 1982) . in such cases the wild-type virus must have a way to throttle the shutoff mechanism. that notion will be pursued in the section on adenoviruses. throughout this section i have tried to point out wrinkles in the data, uncertainties in some popular interpretations, and alternative mechanisms. this critical slant is intended not to minimize the value of the work that has been done, but to stimulate reconsideration of some paradigms that may have been accepted or rejected too quickly. experiments probing the mechanism of host shutoff are difficult. some of the pitfalls and caveats might be stated at the outset. certain techniques that are used to block virus infection at a particular step, in order to define the extent of viral expression that is needed to effect host shutoff, might inadvertently create a new inhibitory mechanism. in the resulting confusion one learns little about the physiological mechanism of inhibition. for example, treatment of poliovirus-infected cells with guanidine not only blocks the synthesis of progeny rna (which is the intended purpose), but also causes double-stranded rna to accumulate to higher-than-normal levels (baltimore, 1969); and double-stranded rna is a potent inhibitor of translation. experiments showing that a temperature-sensitive mutant virus which makes no progeny rna nevertheless shuts off host protein synthesis as effectively as wild-type poliovirus suffer the same defect. the mutant-infected cells accumulate massive amounts of partially double-stranded "replicative intermediates" which are likely to inhibit translation, irrespective of the normal shutoff mechanism (hewlett et al., 1982) . in short, the problem with many experiments is that translation can be inhibited in a variety of ways, and in the process of blocking one pathway, another can be activated. for the same reason, the assumption that the mechanism of host shutoff is the same at high multiplicities of infection as at low multiplicities is untenable. in the case of encephalomyocarditis (emu virus, the effect on host protein synthesis has been shown to differ qualitatively as a function of multiplicity (alonso and carrasco, 1981) . with poliovirus, the familiar statement that guanidine does not prevent host shutoff is true only when the cells are infected at a high multiplicity (helentjaris and ehrenfeld, 1977) . at a normal multiplicity of infection, guanidine does block host shutoff, and therefore it is not clear that viral rna synthesis (which is the guanidine-sensitive step) is uninvolved in the normal mechanism of host shutoff by poliovirus. the specific deficiency or alteration in the translational machinery can sometimes be pinpointed by studying protein synthesis in extracts prepared from virus-infected cells, provided that one appreciates the limitations of that approach. the notion that one can study the mechanism of host shutoff by one virus by using a second virus as a stand-in for host mrna is questionable, because proteins encoded by two differhelentjaris and ehrenfeld (1978) ; nuss et al. (1975) . (2) bienz et al. (1978) . (3) bossart and bienz (1981) ; femandez-munoz and darnell (1976) . (4) celma and ehrenfeld (1974) . (5) h e a l and . (6) etchison etal. (1982) ; a. dasgupta, personal communication. (7) helentjaris and ehrenfeld (1977) . emc uirus in hela cells: (1) jen e t d . (1980) . (2) carrasco and lacal (1983). (3) alonso and carrasco (1981) . (4) jen et al. (1980) . (5) alonso and carrasco f1982b); lacal and c a r r a m (1982). (6) mosenkis et al. (1985) ; a. p. rice etal. (1985) . sindbis and sfv: (1) lachmi and kaiiriiiinen (1977) ; wengler and wengler (1976) . (2) and (3) simizu (1984) . (4) van steeg et al. (1981) ; wengler and wengler (1976) . (5) carrasco and lacal(1983) ; gamy et d. (1979) . (6) van steeg et al. (1981) . (7) simizu (1984) . vsv: (1) lodish and porter (1981) ; mcallister and wagner (1976) . (2) grinnell and wagner (1985) . (3) jaye et al. (1982) ; lodish and porter (1980) ; nisbioka and silverstein (1978a) . (4) lodish and porter (1980) ; otto and lucas-lenard (1980). (5) francoeur and stanners (1978) ; . (6) centrella and lucas-lenard (1982) ; dratewka-kos etal. (1984) . reouirus in l cells: (1) zweerink and joklik (1970) . (2) sharpe and fields (1982) . (3) (1984) . skup and millward (1980) . (7) sharpe and fields (1982) . influenza virus: (1-3) inglis (1982) ; . (4) lazarowitz etal. (1971) . (5) carrasco and lacal (1983) . (6) katze et al. ( , 1986 . adenouirus: (1) castiglia and flint (1983) . (2) babich et al. (1983) ; beltz and flint (1979) . (3) babich et al. (1983) . (4) castiglia and flint (1983) . (6) see text. (7) babiss and ginsberg (1984) . vaccinia uirus: (1) hruby and ball (1981) ; oppermann and koch (1976) . (2) salzman etal. (1964) . (3) cooper and moss (1979) ; rice and roberts (1983) . (4) oppermann and koch (1976) ; rice and roberts (1983) . (5) norrie etal. (1982) . (7) bablanian etal. (1981) . herpes simpler: (1) pereira et al. (1977) . (2) fenwick and walker (1978) ; stenberg and pizer (1982) . (3) stage 1-see text; stage 2inglis (1982) ; nishioka and silverstein (1978b) . (4) silverstein and engelhardt (1979) . (5) fenwick and walker (1978) ; hackstadt and mallavia (1982) . (7) fenwick and walker (1978) ; nishioka and silverstein (1978b) ; read and frenkel (1983) . frog virus 3: (6) cited in mosenkis et al. (1985) . all other entries are from willis et al. (1985) . bthe timing of host shutoff relative to the onset of viral translation is indicated. a capitalized entry in this or any other column identifies the probable major mechanism of host shutoff. "coincident" in capitals means that competition probably underlies host shutoff. cfunctional stability is usually evaluated by the ability of host mrnas, extracted from infected cells, to be translated in a cell-free reticulocyte lysate. dthis column indicates the presence or absence of a temporal correlation between the inhibition of host protein synthesis and the influx of sodium ions that often accompanies virus infection (carrasco and lacal, 1983) . ea change in cap binding protein was postulated because extracts from sfv-infected cells were unable to translate most capped mrnas, with the exception of emc and sfv late 26s mrnas (van steeg et al., 1981 1. although it is true that efficient mrnas like emc and sfv 26 s can be translated without benefit of the m7c cap, it does not follow that cap binding protein(s1 are deficient in every instance where translation of those mrnas persists in the face of an overall decline. efficient mrnas will be selectively translated when any component of the translational machinery is made limiting. the best evidence for this is the ability of both emc and sfv 26 s mrna to be translated in emc virus-infected cells, in which host translation is drastically inhibited by a mechanism that has not been difined, but that clearly does not involve cap binding protein (mosenkis et al., 1985) . wan steeg et al. (1984) have postulated that capsid protein is responsible for host shutoff by sfv, but the evidence is not compelling: the binding of host mrna to ribosomes was only slightly inhibited in fig. 4 of their paper, and the inhibition was a t the level of 80 s rather than 40 s ribosomes. the fact that translation of late viral 26 s mrna was unaffected is not adequate evidence of specificity, since 26 s mrna-by virtue of its high efficiency-would be relatively resistant to any inhibitor, physiological or otherwise. gwith type-2 reovirus in l cells, infection at a multiplicity of infection (moi) of 10 caused no significant decrease in translation; a t mol of 20, translation gradually declined by -40% (sharpe and fields, 1982) . with type-3 reovirus (moi of zo), overall protein synthesis was initially stimulated in both hela and l cells; translation declined later only in l cells (munoz et al., 1985a) . hrecent data do not corroborate an earlier hypothesis concerning inzctivation of a cap-specific translation factor (skup and millward, 1980) . although extracts from reovirus-infected cells translate capped reovirus mrnas poorly, other cap-dependent mrnas, such as globin and tobacco mosaic virus, are translated efficiently in such extracts (lemieux et al., 1984) ; and capped sv40 mrnas are translated in cells coinfected with reovirus (daher and samuel, 1982) . perhaps translation of capped reovirus mrnas is inhibited (artificially) in extracts from infected cells because viral structural proteins, which must be abundant in those extracts, adsorb to the homologous mrnas and sequester them from ribosomes. 'in contrast with most other host mrnas, the synthesis of histone mrnas is inhibited in adenovirus-infected cells (flint et al., 1984) . ]the 55-kda e1b protein probably functions only indirectly to shut off host translation. the protein is required for efficient cytoplasmic accumulation of late viral -as, which might in turn shut off host protein synthesis by competition (see text). proteins from regions e1b and e4 may function as a complex. khost transcripts were stable by hybridization when hela cells were infected in the presence of actinomycin d (rosemond-hornbeak and moss, 1975) but were degraded during productive infection of l cells by vaccinia virus (rice and roberts, 1983) . the second observation seems more pertinent. 'ben-hamida et al. (1983) have purified a component from vaccinia virions that blocks the binding of met-trna to 40 s ribosomes in uitro, but the physiological (in uiuo) mechanism of host shutoff seems to require the expression of viral genes. there is no evidence that eif-2 function is impaired in infected cells. it is possible, however, that some component in the eif-2 cycle is altered in a positive way, i.e., a way that prevents inactivation by eif-2 kinase (whitakerdowling and youngner. 1984) . "it is clear that host translation can be inhibited rapidly in the presence of drugs that preclude the synthesis of viral mrna (moss, 1968) . but it is not clear that the normal shutoff mechanism is at work in such cases (see text). "the virion-mediated rapid shutoff of host translation is not usually seen with herpes simplex type 1. except in vero cells; type 2 virus displays the early shutoff function in all cell types. an important, albeit undeciphered, clue is that type 1 virus interferes with the early shutoff by type 2 virions in doubly infected friend erythroleukemia cells (hill et al., 19851. ent viruses can often be produced simultaneously in cells in which host protein synthesis is suppressed (alonso and carrasco, 1982a,c; otto and lucas-lenard, 1980) . some features of the intracellular environment, such as ionic changes that favor the translation of viral over host mrnas, are inevitably lost when the cells are lysed, and other features are not easily preserved. for example, phosphorylated initiation factors have sometimes been inadvertently restored to normal during their purification (centrella and lucas-lenard, 1982; wong et al., 1982) . phosphorylation of eif-2 has also been missed on occasion because eif-2(ap), retains the ability to function stoichiometrically, and the defect is evident only if one assays for catalytic function (safer, 1983) .6 on other occasions, phosphorylation of eif-2 has been missed because a high concentration of gtp in the lysate masks the functional defect (schneider et al., 1985) . extreme care is needed also to preclude the artifactual modification of initiation factors-by proteolysis, for example-during the preparation of cell-free extracts. the fact that one can reproduce in vitro the preferential translation of viral over host mrnas does not necessarily mean that one is studying the physiological mechanism of host shutoff. if viral mrnas are even slightly more efficient than host mrnas, as is often the case, any manipulation that establishes competition will favor the viral mrnas. one cannot define how competition is established in uiuo by showing that competition occurs in uitro. for example, the fact that translation of vaccinia mrnas is more resistant than host mrnas to inhibition by poly(a) when translation is studied in cell-free extracts from reticulocytes (bablanian and banerjee, 1986; coppola and bablanian, 1983) does not mean that vaccinia virus inhibits host translation by flooding the cytoplasm with short, polyadenylated transcripts. such transcripts are indeed produced in infected cells, but only when drugs are used to block the synthesis of normal viral mrnas (rosemond-hornbeak and moss, 1975) . the aforementioned problem of an experimental manipulation creating a new inhibitory mechanism, rather than exposing the normal mechanism, almost certainly applies here. the tendency to attribute functional significance to foreign agents that cosediment with polysomes should be resisted. everything cosediments with polysomes to some extent. the presence of a trace of ade-6 eif-2, eukaryotic initiation factor 2, is responsible for binding initiator methionyl-trna to the 40 s ribosomal unit, and eif-2(ap) is eif-2 phosphorylated on its a-subunit. when eif-2 is phosphorylated, the reaction in which gdp is exchanged for gtp fails. that reaction is mediated by an accessory protein called gef, which becomes trapped in an inactive complex with eif-2(ap). because the pool size of gef is small, phosphorylation of only 30% of the eif-2 pool can completely inhibit translation (safer, 1983; siekierka et al., 1984) . 25 1 novirus va-rna in the polysome region of sucrose gradients (schneider et al., 1984) , for example, is almost certainly unrelated to the function of va-rna. it is common to find viral capsid proteins stuck to ribosomes, and it is wise to treat such contamination as contamination, until it is proven otherwise. the known and suspected mechanisms by which translation of viral mrnas is facilitated, usually to the disadvantage of host mrnas, fall into four categories. 1. competition may be suspected when the decline in host protein synthesis and the onset of viral protein synthesis coincide. on the other hand, competition is an insufficient explanation when host protein synthesis is severely inhibited before the onset of viral translation, as occurs with poliovirus, herpes simplex virus, and frog virus 3. often competition is exacerbated by a decline in the overall translational capacity, which may be brought about by changes in the ionic environment or in the translational machinery. when initiation is limiting, most mrnas accumulate in small polysomes, the size of which increases upon exposure to a low concentration of cycloheximide. (cycloheximide slows elongation, thus causing the number of ribosomes to increase on mrnas that were previously limited at the initiation step.) the characteristic shift in polysome size upon exposure to cycloheximide is seen, for example, in cells infected by vsv (jaye et al., 1982) or adenovirus (perlman et al., 1972) . the competition between host and viral mrnas that takes place in uiuo is sometimes not maintained when the translation of endogenous mrnas is studied in extracts from infected cells, probably because the concentrations of critical components change during the preparation of such extracts. 2. inactivation of a normal component of the translational machinery. the resulting deficiency enables only a subset of mrnas, mostly viral, to be translated. the hallmark of this mode of regulation is the ability to restore translation to cell-free extracts by adding back the missing factor, in practice, this is not as easy as it sounds. there is evidence for inactivation of initiation factor eif-2 in several virus systems, as noted in table i . alterations in the initiation factor that mediates the translation of capped mrnas have been postulated for several viruses (table i) , but the story seems credible only in the case of poliovirus, which is described below. identifying characteristic here is that extracts from infected cells cannot be reactivated by the addition of normal initiation factors, but can be reactivated by washing the ribosomes to remove the inhibitor. based on these criteria, pensiero and lucas-lenard (1985) have postulated the production of an inhibitor during mengovirus infection. because mengovirus and host mrnas are equally sensitive to the inhibitor in cell-free extracts, one must invoke competition (for the residual functional ribosomes) t o explain the selective persistence of viral translation in uiuo. this seems justified in view of the extraordinary efficiency of mengovirus mrna when translation is carried out in uitro under conditions of competition (abreu and lucas-lenard, 1976) . until the postulated inhibitor has been identified, however, we cannot be certain that mengovirus belongs in category 3. the hallmark here is that viral mrnas should be translated more efficiently in cell-free extracts from infected than from uninfected cells. frog virus 3 meets this criterion (raghow and granoff, 1983) . it is possible that some other animal viruses alter the translational machinery in a "positive" way.7 the best evidence t o date comes from plant viruses, however. a genetic analysis of temperature-sensitive mutants of alfalfa mosaic virus strongly suggests that rnas-1 and -2 encode or induce a factor that facilitates the translation of coat protein from rna-4 (huisman et al., 1985) . extrapolating that mechanism to brome mosaic virus would explain why rna-4 fails to synthesize coat protein when it is injected (without rnas-1, -2, and -3) into barley protoplasts (kiberstis et al., 1981) . unfortunately, cellfree systems from infected plant cells are not available to test the hypothesis. the aforementioned hints are only hints. no virus has yet been proved to produce a new or alter an old translational factor in a way that specifically promotes its own translation. since competition is the most common mechanism of translational regulation in virus-infected cells, that topic merits more attention. there are at least three variations on the theme. although the overall ability to translate poliovirus mrna is about the same when cell-free systems are reconstituted with factors from infected or uninfected cells (brown and ehrenfeld, 1980) , there is a qualitative difference in the selection of initiation sites when factors from infected cells are used (brown and ehrenfeld, 1979) . other experiments support the idea that poliovirus (bernstein et al., 1985) as well as vaccinia (moss and filler, 1970) and human t-lymphotropic virus type i11 (rosen et al., 1986) produce something that enhances the synthesis of viral proteins. the enhancing substance could viral translation 253 in l cells infected by reovirus type 2, host protein synthesis is dramatically shut off. the elegant genetic studies of sharpe and fields (1982) revealed that the s4 gene, which encodes the major capsid protein u3, is responsible for the inhibition. the effect of u3 might be indirect, inasmuch as the same gene product is responsible for inhibiting host rna synthesis. although host shutoff by type 3 reovirus in sc-1 cells is less dramatic than with type 2 virus in l cells, the mechanism of type 3 shutoff is better understood due to the careful quantitative studies of thach and colleagues (walden et al., 1981) . their conclusion was rather surprising: the intrinsic translational efficiency of reovirus mrnas is not higher than that of host mrnas, but rather, reovirus translation dominates because viral mrnas accumulate in massive amounts-up to 45% of the total mrna in the cell! the evidence that reovirus mrnas initiate translation less efficiently than most host mrnas is twofold: (1) the size of reovirus polysomes is smaller than host polysomes that code for proteins of comparable size; and (2) whereas a low concentration of cycloheximide reduces the synthesis of host proteins (which is the result expected for mrnas of "normal" efficiency), the translation of reovirus proteins is actually enhanced by a low concentration of cycloheximide. whereas reovirus mrnas appear to be less efficient than most host mrnas, vsv mrnas are probably translated as efficiently as host mrnas, but not more so. competition is simply proportional to the concentration of viral mrnas in the cytoplasm (lodish and porter, 1981) ,8 and vsv and host mrnas that encode the same-sized proteins are on polysomes of the same size (lodish and porter, 1980) . in some cell lines infected by some strains of vsv, a portion of the eif-2 pool seems to be inactivated (centrella and lucas-lenard, 1982; dratewka-kos et al., 1984) . although that would intensify the competition, it is obvious that lowering the eif-2 level per se cannot explain the selective inhibition of host translation. selective shutoff requires that viral mrnas be more abundant than host mrnas, or more efficient, or be a virus-specific translation factor, or a protease inhibitor that stabilizes viral proteins, or a nuclease inhibitor, or something else. recent evidence indeed suggests that vaccinia encodes a function that protects late viral mrnas against degradation (pacha and condit, 1985) . both. the aforementioned experiments argue against vsv mrnas being unusually efficient, but other experiments have been taken as evidence for the contrary view. because vsv mrnas are more resistant than host mrnas to hypertonic stress, nuss et al. (1975) have suggested that the viral mrnas are intrinsically more efficient. their interpretation seems reasonable, but it must be carefully circumscribed. if high salt exacerbates some deleterious feature in the mrna (such as secondary structure) to the point where it becomes inhibitory, then the hierarchy of mrna strengths that one observes under hypertonic conditions might be irrelevant to normal growth condition^.^ the progressive inhibition of host protein synthesis during infection by vaccinia virus is probably due to competition, in proportion to the concentration of each mrna. viral mrnas are not apparently more efficient than host mrnas (cooper and moss, 1979; . degradation of host mrnas (table i ) and the massive synthesis of viral transcripts probably tip the balance in favor of viral protein synthesis. have suggested that differential association of mrnas with the cytoskeleton might also play a role, but that is a difficult hypothesis to test. in contrast with reovirus and vsv, the concentration of emc virus mrna in infected cells may be too low for simple competition to effect the observed switch from host to viral translation, even though emc mrna is translated more efficiently than host mrnas both in uiuo (jen et al., 1978) and in vitro (golini et al., 1976; svitkin et al., 1978) . in view of the overall decline in translation that begins 3 hours postinfection, however, the idea that emc virus mrna outcompetes host mrnas for the low, residual translational capacity seems reasonable. the overall decline is most likely due to an influx of monovalent cations, since the two events are temporally correlated (lacal and carrasco, 1982) . host translation is restored when emc virus-infected cells are shifted to hypotonic medium carrasco, 1981, 1982b1 , and excess salt, sufficient to inhibit the translation of host mrnas in uitro, dramatically stimulates the translation of emc rna (carrasco and smith, 1976 ). 9recall that, although reovirus mrnas are not more efficient than host mrnas in unperturbed cells, reovirus translation, like that of vsv, dominates when cells are subjected to hypertonic stress (nuss et al., 1975) . in other studies, the creation of a hairpin (ag -30 kcalimol) within the 5'-noncoding region of preproinsulin mrna impaired translation only in hypertonic medium; the hairpin did not inhibit translation under normal culture conditions (kozak, 1986b ). a similar mechanism might mediate the switch from host to viral translation during infection by alphaviruses (see sindbis and sfv, i.e., semliki forest virus, in table i) , since the influx of sodium ions exactly coincides with the overall decline in protein synthesis. the magnitude of the ion influx remains controversial (gray et al., 1983; munoz et al., 1985b) . translation of sfv mrna is more resistant than host protein synthesis to hypertonic conditions (garry et al., 19791 , but the resistance is not as dramatic as with emc virus (alonso and carrasco, 198213 . the notion that alphaviruses inhibit host translation by competition seems viable even if something more than enhanced permeability to monovalent cations is needed to explain the overall decline. the fact that sfv mrna can be translated in emc-infected cells (alonso and carrasco, 1982~1 , in which the overall translational capacity is very low, identifies sfv late 26 s mrna as an efficient message. consistent with the competition hypothesis, the time of host shutoff coincides with the production of viral mrna (lachmi and kaariainen, 1977) and the severity of inhibition correlates with the yield of viral rna in mutant-infected cells (atkins, 1976) . polysomes containing sfv (wengler and wengler, 1976) or emc virus mrna do not increase in size upon exposure to cycloheximide, suggesting that those mrnas are efficient enough to be fully loaded with ribosomes even when the overall translational capacity is low. influenza virus mrnas are translated with extraordinarily high efficiency i n uitro (katze et al., 1986) . because the shutoff of host protein synthesis coincides with the onset of influenza virus protein synthesis and there is no overall decline in translation, simple competition would seem adequate to explain the switch from host to viral translation. in the case of adenovirus, competition is probably exacerbated by a reduction in functional eif-2 levels. these issues are discussed in more detail in section ii1,e. in some virus systems, competition might dictate the switch from synthesis of early to late viral proteins. picornaviruses, rhabdoviruses, and influenza virus are uninteresting in this regard, as they display little or no temporal control over protein synthesis. the existence of a temporal switch is questionable for reoviruses, but all of the other entries in table i , as well as the papovaviruses, show a striking earlyto-late transition. in every case, the switch is effected primarily a t the level of transcription: the mrnas that encode late proteins are not synthesized until late. in several cases, however, early mrnas persist in the cytoplasm at late times, and some form of translational regulation seems to limit their expression (hruby and ball, 1981; johnson and spear, 1984; lachmi and kaariainen, 1977; vassef et al., 1982) . with black beetle virus that phenomenon can be attributed to competition, because translation of the late mrna predominates over early mrna in cell-free extracts under conditions of competition (friesen and rueckert, 1984) . the same explanation probably holds for alphaviruses. on the other hand, late vaccinia virus mrnas do not appear to be more efficient than early viral mrnas (cooper and moss, 1979; oppermann and koch, 1976) . instead, degradation of some early vaccinia transcripts (hruby and ball, 1981) might be part of the switching mechanism. the translation of early mrnas could be further reduced by the accumulation of "anti-early mrna" (boone et al., 19791 , which could inhibit translation much as antisense rna does in other experimental systems (izant and weintraub, 1985) . lo with baculoviruses, temporal switching involves the sequential activation of upstream promoters, such that the small, early mrnas are replaced by progressively longer overlapping transcripts (friesen and miller, 1985) . the resulting relegation of early protein coding sequences to the 3' ends of late transcripts probably prohibits their translation. promoter switching late in sv40 infection also generates forms of mrna from which t antigen is translated inefficient1y.l thus, although transcription plays the dominant role, translational mechanisms-involving competition or other ploys-contribute to the temporal switch in expression of viral genes in some systems. the current thinking is that poliovirus selectively shuts off host protein synthesis by inactivating a 220-kda protein (~2 2 0 ) which is a subunit of the initiation factor that mediates the translation of capped mrnasll because the 5' end of poliovirus mrna is uncapped, inac-10 few systems other than vaccinia show much potential for regulating translation by "hybrid arrest." complementary transcripts accumulate in the nuclei of many virusinfected cells, but the complementary sequences are usually edited from cytoplasmic mrnas. in the case of adenoviruses, for example, where transcription switches periodically from one dna strand to the other, the 3' ends of the juxtaposed mature mrnas rarely overlap (lemoullec et al., 1983) . the 3' ends of papovavirus early and late mrnas do characteristically overlap, however. 11 although the proteins that can be cross-linked to the m7g cap have a disturbing tendency to change from year to year, two proteins in mammalian cells that reproducibly cross-link are p24-cbp and p46-cbp. p220 is not a "cap binding protein" inasmuch as it does not cross-link to the cap, but p220 does copurify with p24-cbp and p46-cbp. "he aggregate, called eif-4f, is considered by most people to be the functional "cap binding factor." the functions of cap binding proteins have been reviewed by shatkin (1985) . tivation of the cap binding factor should not impair the translation of viral mrna. the idea is appealing because it is straightforward, but some of the supporting data are less so. the experiment that gave birth to the hypothesis was provocative. using an antiserum against initiation factor eif-3, etchison et al. (1982) showed by immunoblotting that p220 is clipped during the first few hours after infection of hela cells by poliovirus. because affinitypurified antibodies against p220 recognized a protein of the same size in some preparations of cap binding factor, the working hypothesis was that cleavage of p220 inactivated the cap-binding initiation factor. indeed, an activity from uninfected cells that was subsequently purified, based on its ability to restore translation to poliovirus-infected cell-free extracts, contained p220 and the cap binding proteins. these experiments are described below in more detail. this is the most promising explanation to come forth, but there are some irregularities and many lacunae in the supporting data. 1. there is a discrepancy between the kinetics of degradation of p220 and the kinetics of host shutoff (etchison et al., 1982) . the same anomaly occurs during infection by rhinovirus, which degrades p220 in a manner similar to poliovirus (etchison and fout, 1985) : the rate of translation is still half-maximal a t the time when p220 disappears from the polyacrylamide gels. in many of the experiments carried out with cell-free extracts, the question of timing was disregarded, and cells were routinely harvested 3 hours postinfection (lee and sonenberg, 1982; lee et al., 1985a) , which is well beyond the point when host translation is precipitously shut off. 2. the extent of cleavage is difficult to evaluate quantitatively. it seems dangerous to accept the recommendation of bernstein et al. (1985) to focus on the accumulation of the 115-kda cleavage products without also monitoring the disappearance of p220, because cleavage need not always be arrested at the 115-kda level. in some experiments the concentration of cleavage products in immunoblots from infected cells greatly exceeded the concentration of intact p220 in uninfected cells (bernstein et al., 1985) . 3. a p220 cleavage pattern qualitatively similar to that which occurs in infected cells, although not nearly as extensive, is evident in some extracts from uninfected cells (bernstein et al., 1985; fig. 5 in lee et al., 1985a) . because the link between degradation of p220 and virus infection is not tight, it was not surprising to find that the virus-encoded protease 3c is not responsible for cleaving p220 (lee et al., 1985b; lloyd et al., 1985) . it would seem wise to include a spectrum of protease inhibitors during the preparation of extracts. phenylmethylsulfonyl fluoride is the only one routinely used, at concentrations ranging from 5 mm (which is adequate; etchison et al., 1982) to 1 mm (bernstein et al., 1985) or 0.2 mm (lee and sonenberg, 1982) . one is reminded of the old excitement concerning "processing" of sv40 t antigen (ahmad-zadeh et al., 1976) which turned out to be an artifact of extraction (smith et al., 1978) . 4. in uninfected hela cells, the concentration of p24-cbp is 10-fold lower than p220 (duncan and hershey, 1985a,b) . [the concentration of p24-cbp is also low in reticulocytes (hiremath et al., 1985) .1 if p24 and p220 (together with p46) function as the cap binding complex, large changes in the p220 pool-although easy to detect by immunoanalysis-are unlikely to alter the rate of translation; but small changes in the pool of p24, which might go undetected with immunological or biochemical probes, would significantly impair translation. 5. a mutant of poliovirus called hf121 has been described in which the synthesis of viral rna is normal in cv-1 cells, but viral protein synthesis is inefficient, host translation is inhibited more slowly than usual, and p220 is not rapidly cleaved (bernstein et al., 1985) .12 (the phenotype of hf121 in hela cells, which are a more natural host than cv-1 monkey cells, is more complex. the synthesis of viral rna is greatly reduced and all protein synthesis, host and viral, is inhibited very early, again without the concomitant cleavage of p220.) the authors argue cogently that wild-type poliovirus appears to encode a function, absent from hf121, that promotes (or "avoids the early inhibition of") viral translation, and they argue less cogently that hf121 is translated poorly as a consequence of the failure to selectively inhibit host translation. to me, the second postulate seems redundant. lack of the putative positive factor would be sufficient to account for the poor translation of viral mrna, and the failure to inhibit host translation with normal kinetics could as likely be the result of ineffkient viral translation as the cause.i3 if the slow inhibition of translation by 12 although cleavage of p220 is not detectable at all in hela cells infected by mutant hf121, cleavage products are clearly evident in cv-1 cells a t 5 hours postinfection (fig. 8a, lane 7, in bernstein et al., 1985) . that is later than normal, and the cleavage is less extensive than normal, but some cleavage does occur. 13 one could argue similarly that cleavage of p220 in wild-type infected cells is the consequence of the abundant accumulation of viral proteins, rather than a precondition. the issue might be resolved by treating wild-type infected cells with guanidine, which allows only limited synthesis of viral proteins while host translation is inhibited with the usual rapid kinetics. it would be informative to know whether p220 undergoes cleavage under those circumstances. hf121 in cv-1 cells is a delayed version of the normal shutoff mechanism, then cleavage of p220 must not be central to the normal shutoff mechanism. the authors argue, to the contrary, that shutoff by hf121 is mechanistically different, inasmuch as the inhibition affects both host and viral mrnas in hf121-infected cells, whereas host translation is preferentially inhibited in wild-type-infected cells. however, selective stimulation of viral translation (mediated by the product that is defective in hf121) superimposed on a general inhibition of translation, would mimic selective inhibition. the authors contend that the ability of guanidine to block host shutoff by hf121 distinguishes it from the normal shutoff mechanism, but the experiment (in cv-1 cells) was done without testing wild-type virus in parallel, as could have been done by adding guanidine at the start of the infection rather than after 3 hours. 6. the assumption that tobacco mosaic virus, sindbis virus, and vsv mrnas are appropriate stand-ins for host mrna in the restoring assay is questionable rose et al., 1978; tahara et al., 1981) . in cells singly infected by vsv, viral mrnas are translated in preference to host mrnas under some conditions (nuss et al., 1975) ; thus, vsv mrnas are not equivalent to most host mrnas. when cells are doubly infected with poliovirus and sfv (which is akin to sindbis), or with poliovirus and vsv, conditions can be found that allow the simultaneous translation of poliovirus mrna and the capped mrnas of vsv or sfv (alonso and carrasco, 1982a) . thus, the factor that restores to poliovirus-infected extracts the ability to translate vsv or alphavirus mrnas might not be sufficient to restore the translation of most host mrnas. globin is the only cellular mrna that has been shown to work in the restoring assay (edery et al., 19841 , and its translational efficiency resembles viral mrnas more than the average cellular mrna. it would be reassuring to omit the usual micrococcal nuclease pretreatment of lysates, and show that the addition of cap binding factor to poliovirus-infected cell-free extracts restores the translation of authentic endogenous host mrnas. the association of p220 with p24-and p46-cbps does not prove that p220 is a necessary component of the complex. in early studies, preparations of p24-cbp that lacked the p220 subunit did preferentially stimulate the translation of capped mrnas in hela cell-free extracts (tahara et al., 1981; sonenberg et al., 1980) . those results were considered wanting because p24-cbp failed to reproducibly restore translation to extracts from poliovirus-infected cells, whereas an aggregate of p220, p46, and p24 could restore (tahara et al., 1981) . but there is no reason to reject the aforementioned demonstration that p24-cbp by itself does stimulate in uninfected systems. using the restoring assay to define the structure of cap binding factor would be acceptable only if one knew that cap binding factor was deficient in infected-cell extracts. grifo et al. (1983) showed that translation of globin mrna was stimulated by the p22o-p46-p24 aggregate, even when the system was saturated with p24-and p46-cbps. those data prove only that the system which they reconstituted from partially purified subfractions of a reticulocyte lysate was deficient in p220; the data do not prove that p220 is an essential component of cap binding factor. (indeed, translation of the uncapped mrna from satellite tobacco necrosis virus was stimulated to the same extent as capped globin mrna.1 the function of p220 would be clearer if one could show that antibodies against p220 inhibit the function of cap binding factor. such experiments have not been reported. indeed, the immunological evidence is far from convincing even for the original p24-cbp. a monoclonal antibody "directed against cap binding proteins" was shown to inhibit the translation of capped mrnas, but the antibody reacted with higher molecular weight proteins and not with p24-cbp (sonenberg et al., 1981) . the claim that the higher molecular weight polypeptides were related to p24-cbp no longer seems valid, because polyclonal antibodies obtained recently against p24-cbp react only with that polypeptide (hiremath et al., 1985) . in extracts from uninfected hela cells, p220 copurifies to some extent with both cbps and eif-3 (etchison et al., 1982) . whereas it is known that p220 restores translation to poliovirus-infected extracts when it is introduced in association with cap binding proteins, it is not known whether p220 would also enhance were it introduced in association with eif-3. in several studies, eif-3 failed to restore translation to extracts from poliovirus-infected cells, but it was usually tested on an equal weight basis, vis-a-vis the other initiation factors (table iv in grifo et al., 1983; rose et al., 1978) . because eif-3 is so massive, it must be tested on an equal molar basis. an experiment which intended to show the eif-3 from poliovirusinfected cells is fully functional failed to prove the point, because the assay for eif-3 was carried out in the presence of cap binding factor from uninfected cells (etchison et al., 1984) . the exogenous cap binding factor may have contributed a component (such as p220) which was necessary for, and absent from, infected-cell eif-3. the assay would have been more meaningful had an uncapped mrna been used, thus allowing the function of eif-3 to be evaluated without the necessity of adding cap binding factor. whether p220 should be considered a component of eif-3 or of the cap recognition factor involves more than semantics. whereas inactivation of cap binding factor would be sufficient to explain the selective inhibition of host translation, eif-3 is apparently needed for translating all mrnas; were eif-3 activity low, poliovirus would have to outcompete host mrnas for the residual activity. casting a wider net might identify other components that are involved in host shutoff by poliovirus. a few candidates have been ruled out. initiation factors eif-4a and eif-4b, for example, appear to be unaltered (duncan et al., 1983) . the normal association of host mrnas with the cytoskeleton is disrupted shortly after infection by poliovirus (lenk and penman, 1979) . whether that is the cause, or the effect, or is unrelated to inhibition of host translation remains unclear. such a dramatic effect seems unlikely to be gratuitous, but in other systems disruption of the cytoskeleton does not preclude all translation (welch and feramisco, 1985) . follow-up studies in the poliovirus system have not significantly extended penman's original, provocative observation. when bonneau et al. (1985) infected cv-1 cells first with vsv (which does not dissociate host mrnas from the cytoskeleton) and then superinfected with poliovirus, translation of vsv g and m proteins was inhibited and those mrnas were released from the cytoskeleton; unfortunately, it was not shown that vsv n and ns mrnas, which continued to be translated, remained bound to the cytoskeleton. the conclusion that translation requires association with the cytoskeleton hardly seems warranted. carrasco has suggested that the increased permeability of virusinfected cells to monovalent cations might mediate the switch from host to viral translation (carrasco and lacal, 1983; carrasco and smith, 1976) . when infected cells are incubated in medium containing sufficient excess nacl to inhibit the translation of most other proteins, poliovirus translation is fairly resistant (alonso and carrasco, 1982a; nuss et al., 19751 , but the resistance is not as striking as with emc virus (alonso and carrasco, 1982b) . the stimulation of in uitro translation by high salt is also less obvious with poliovirus mrna than with some other picornaviruses (bossart and bienz, 1981) . in the natural course of infection by poliovirus, the precipitous decline in host translation occurs within the first 2 hours, prior to the observed increase in intracellular sodium ions (nair et al., 1979) . moreover, the synthesis of cellular proteins cannot be reactivated by incubating poliovirusinfected cells in hypotonic medium (alonso and carrasco, 1982b) , a manipulation that works beautifully with emc virus. thus, hypertonicity does not appear to underlie the shutoff of host protein synthesis by poliovirus. morrow et al. (1985) made the astonishing discovery recently that the host-encoded kinase that is responsible for phosphorylating eif-2 binds to and mediates the replication of poliovirus rna. although that seems about as auspicious as a sheep shaking hands with a wolf, one can think of ways to rationalize such a dangerous move. if the pool of viral rna that serves as template for replication has to be kept free of ribosomes, for example, the presence of eif-2 kinase in replication complexes could help by phosphorylating the local pool of eif-2. indeed, eif-2 might become globally phosphorylated in infected cells, and the resulting eif-2 deficiency could contribute to the inhibition of host protein synthesis. whereas older studies suggested that eif-2 was not deficient in polio-infected cells (brown and ehrenfeld, 1980; helentjaris and ehrenfeld, 1978) , the translation of heterologous mrnas in infected-cell extracts was restored to a limited extent by the addition of eif-2 (rose et al., 1978) -an effect that the authors chose to ignore. asim dasgupta has reopened the question, and his careful measurements reveal extensive phosphorylation of eif-2(a) in poliovirus-infected cells (personal communication). the adenovirus system has generated considerable excitement recently because genetic manipulations, pioneered in shenk's laboratory, have revealed a regulatory mechanism that is novel, and yet connects to the extensive older literature on inactivation of initiation factor eif-2. the focal point is a small virus-encoded rna called va-rna,. thimmappaya et al. (1982) found that, in cells infected by an adenovirus mutant that produced no va-rnai, late viral mrnas were synthesized, processed, and transported, but failed to be translated. in the absence of va-rna,, translation was blocked at the level of initiation (schneider et al., 1984) and the defect was ultimately localized to eif-2. overwhelming evidence now supports the hypothesis that, in the absence of va-rna,, a kinase becomes activated that phosphorylates, and thus inactivates, eif-2 schneider et al., 1985; siekierka et al., 1985) . eif-2 kinase (one of the enzymes involved in the antiviral action of interferon; see lengyel, 1982) exists in uninfected hela cells in an inactive state, and is apparently activated by double-stranded rna that accumulates in infected cells as a by-product of adenovirus transcription (o'malley et al., 1986) . l4 the exact mechanism by which va-rnai blocks the action of eif-2 kinase is not yet known. an intriguing scenario can be extrapolated from a model that was proposed by rosen et al. (1981) in another context. their model proposes that high molecular weight double-stranded rna activates and targets eif-2 kinase: because both the kinase and eif-2 have binding sites for dsrna, high molecular weight dsrna could link the two proteins.15 by virtue of its small size, va-rna, might be able to bind to eif-2 or to eif-2 kinase, but not simultaneously to both. va-rnai would thus block the phosphorylation of eif-2 much as a monovalent hapten blocks antigen-antibody interactions. the proposal rationalizes the known properties of va-rna,: its small size (about 160 nucleotides), doubled-stranded structure (monstein and philipson, 19811 , and the high concentration that is required to confer protection. whether the double-stranded regions of va-rnai are crucial for its function is not yet clear. have mutated va-rna, and found that extensive regions could be deleted without affecting biological activity, although certain other mutations were deleterious. further experiments will be needed to pinpoint the essential region(s) in va-rnai. a second adenovirusencoded species called va-rnai, rescues translation far less efficiently than va-rna, (thimmappaya et al., 19821 , and va-rnaii appears less extensively base paired (mathews and grodzicker, 1981) . in addition to its proven protective effect on eif-2, it has been suggested that va-rna, might interact directly with viral mrnas to promote their translation (kaufman, 1985; schneider et al., 1984; svennson and akusjarvi, 1985) . a sequence-specific interaction seems unlikely, however, because the small rnas encoded by epstein-barr virus (which are related to adenovirus va-rnas by size but not sequence) can substitute to some extent for va-rnai (bhat and thimmappaya, 1983, 19851 , and the facilitating effect of va-rna, extends 14 a virus that is protected (by va-rna or some other mechanism) from the deleterious effects of its own symmetrical transcription process would also have some resistance to interferon. an interesting story along these lines is emerging with vaccinia virus (rice and kerr, 1984; whitaker-dowling and youngner, 1984) . 15 sen et al. (1978) showed that, once kinase has been activated by binding dsrna, incubation with ribonuclease i11 does not abolish the ability of kinase to phosphorylate histone h1 (which was chosen as a convenient substrate), but neither the extent of trimming by the nuclease, nor the activity of the trimmed kinase on eif-2, were determined. thus, the experiment does not contradict the targeting hypothesis for dsrna. not only to late adenovirus mrnas that carry the standard tripartite leader, but also to early adenovirus mrnas and late mrnas with a truncated version of the tripartite leader (svensson and akusjarvi, 19841, adeno-associated virus mrnas (janik et al., 19821, and various heterologous mrnas (svensson and akusjarvi, 1985) . the protective effect of va-rna, and the shutoff of host translation in adenovirus-infected cells might be two aspects of a single mechanism. if one postulates that the production of va-rna, by wild-type adenovirus is sufficient to protect only a portion of the eif-2 pool, the switch from host to viral protein synthesis could occur because late adenovirus mrnas outcompete host mrnas for the small residual translational capacity. the hypothesis that competition occurs during the late stage of infection by adenovirus is not entirely ad hoc. the overall translational capacity is low late in the infection (castiglia and flint, 1983) ; a portion of the eif-2 pool is phosphorylated (m. mathews, personal communication) ; polysomes are small and their size increases in response to a low dose of cycloheximide (perlman et al., 1972) ; and the decline in host translation correlates with the temporal onset and magnitude of late viral translation (castiglia and flint, 1983) . every mutation that has been shown to prevent host shutoff also prevents the cytoplasmic accumulation of late viral mrnas (babiss et al., 1985; halbert et al., 1985) . an interesting set of experiments by logan and shenk (1984) can be rationalized in terms of competition during the late stage of infection. they observed that transposition of the late tripartite leader to the early e1a genes had no effect on the efficiency of translation of e1a products at early times, but significantly enhanced the translation of e1a mrnas at late times. this is understandable if there is no competition early in the infection, allowing efficient and inefficient mrnas to be translated equally well. the facilitating effect of the tripartite leader would become evident only late in the infection, when eif-2 has been partially inactivated and competition has set in. one might think along similar lines to explain the surprising ability of influenza virus mrnas to be translated in adenovirus-infected cells . in wild-type adenovirus-infected cells, in which host protein synthesis is drastically reduced, both adenovirus and influenza virus mrnas are translated efficiently. in cells infected by d1331, a deletion mutant that produces no va-rna,, adenovirus proteins are translated inefficiently, as noted above, but influenza virus proteins are still synthesized in abundance. despite that striking observation, there is little support or necessity for the notion that influenza virus establishes its own translational system. a simpler explanation is that the very low capacity for protein synthesis that persists in the absence of va-rna, is sufficient for the translation of influenza virus mrnas.16 for that explanation to be correct, influenza virus mrnas would have to be translated with extraordinary efficiency, and that prediction has recently been confirmed (katze et al., 1986) . what makes all this so intriguing is that the 5' ends of influenza virus mrnas, which presumably dictate their high translational eficiency, are derived from host mrnas (krug, 1981) . it appears as if the viral capspecific endonuclease (which selects the cellular mrnas that will serve as donors) is biased toward the same features that facilitate translation. indeed, that deduction has been verified directly, at least in vitro. bouloy et al. (1978 bouloy et al. ( , 1980 found that p-globin mrna, which is translated more efficiently than a-globin mrna, is also a more efficient primer for influenza transcription; and alfalfa mosaic virus rna-4, which translates in vitro with extraordinary eficiency, is the best known primer for influenza transcription. [from the fact that mrnas with 2'-o-methyl groups in the penultimate position of the cap function better than monomethylated caps as primers for influenza virus transcription (bouloy et al., 1980) , one is tempted to suggest that 2'-0methyl groups enhance translation, although there is little direct evidence for that view!] if the selection of primers in infected cells follows the pattern that is seen in vitro, the influenza virus takeover scheme is indeed remarkable: the most efficient cellular mrnas would be sacrificed to construct viral mrnas that ips0 facto translate most efficiently. a few other possibilities for regulating translation in virus-infected cells are discussed briefly below. none of these topics is well understood at present, and the musings should be considered little more than that. cauliflower mosaic virus seems to be the only case in which the ability of eukaryotic ribosomes to reinitiate is fully exploited to produce several full-length proteins from one mrna. the structure of a few 16 katze et al. (1986) have shown that influenza virus partially suppresses the activation of eif-2 kinase, and they suggest that this underlies the ability of influenza virus to replicate in cells infected by adenovirus mutant d1331. that interesting hypothesis would be stronger if it could be shown that influenza virus replicates even in cells infected by the adenovirus double mutant (vai -vaii -), and if superinfection with influenza virus could be shown not just to reduce the level of activated kinase, but to increase the level of functional eif-2. other viral and cellular mrnas leads one t o predict that reinitiation is necessary for ribosomes to reach the major protein coding sequence, but the upstream open reading frames (orfs) in such mrnas are characteristically short. in some cases, however, the small peptides encoded near the 5' end of the message might be biologically important. genetic studies indicate that this is certainly the case with the agnogene product of sv40 (margolskee and nathans, 1983) . in contrast, the three peptides encoded within the avian retrovirus leader sequence probably are not functional because there is little conservation of amino acid sequences among virus strains (hackett et al., 1986) . in retrovirus mutants that lack most of the leader sequence, the only known deficiency is the absence of a cis-acting packaging signal (mann et al., 1983; nishizawa et al., 1985) . comparison of different strains of poliovirus reveals that the number of upstream aug codons varies and the coding properties of the small orfs are not conserved (toyoda et al., 1984) . upstream minicistrons that do not encode anything interesting might nevertheless be important for regulation. several possibilities come to mind for retroviruses. the least interesting idea is that upstream aug codons accumulate, not from design, but from defaultbecause the deleterious effects on translation can easily be compensated by using efficient transcription signals to mass-produce retrovirus mrnas. the opposite view is that upstream aug codons are deliberately retained to throttle the synthesis of a protein that would be harmful if overproduced (tarpley and temin, 1984) . while that seems a reasonable ploy to use for oncogenes, it makes little sense when extended to viral structural genes. a third possibility derives from the observation that reinitiation usually is not 100% efficient. with preproinsulin constructs, for example, in which the efficiency of reinitiation is routinely 20% (kozak, 1984b) , one might ask what the remaining 80% of the ribosomes are up to. one scenario is that, after 80 s ribosomes have moved through the 5'-proximal orf, 80% of the 40 s subunits detach at the terminator codon while the rest remain on the message, resume scanning, and reinitiate at the second aug codon. a more interesting possibility is that all 40 s subunits remain bound and resume scanning, but only 20% reinitiate at the closest aug codon, perhaps because the codon-recognition step in inefficient in the absence of met-trna,, cap binding proteins, and/or other initiation factors-all of which were presumably released at an earlier step. [we do not know the precise sequence of events during initiation, but it seems likely that the factors that mediate the binding of met-trna, and mrna to the 40 s ribosomal subunit are released prior to or during the joining of the 60 s subunit at the first aug codon (moldave, 1985) .] if the factor-deficient 40 s subunits that are unable to reinitiate at the second aug codon eventually become competent, they might reinitiate father downstream. thus, the effect of an upstream minicistron could be to loosen the process of initiation in a way that permits ribosomes to reach otherwise inaccessible internal aug codons. there is no evidence for this, as yet. we know only that reinitiation at the closest aug codon (following a terminator codon) is less than 100% efficient. yet another way in which ribosomes might gain access to internal aug codons, even in a message in which the major open reading frame initiates with a "strong" aug codon, relies on the presence of weak, out-of-frame initiator codons in the retrovirus leader sequence and the ability of ribosomes to reinitiate. this hypothetical scheme is best illustrated by using as an example an avian retrovirus mrna that encodes the e m glycoprotein (fig. 2) . katz et al. (1986) have studied the effects of mutations in the leader region of this mrna, using as an a hypothesis whereby minor initiation sites in the leader sequence of retroviruses create a shunt that directs ribosomes to internal initiation sites. the diagram represents a subgenomic mrna that encodes the enu protein of avian leukosis virus (katz et al., 1986) . messenger rna is represented by a wavy line, the pathway followed by 40 s ribosomal subunits is shown above the mrna, and the pathway of 80 s ribosomes is shown below the mrna. a solid black line traces the pathway followed by most 40 s subunits: they scan from the m7g cap to the start of the enu coding sequence, marked "major start site," where a 60 s subunit joins and translation begins. (some 40 s subunits will stop and initiate at three upstream aug codons, but in each instance there is a nearby terminator codon, enabling ribosomes to reinitiate. thus, the upstream aug codons are irrelevant for the present discussion and are not shown.) of more significance are the many nonstandard codons (gug, uug, etc.) that lie in the standard context for initiation. such codons occur frequently in the -1 reading frame which is open (in the functional ev-2 viral genome) over a stretch of about 200 nucleotides preceding the major enu start site; the open -1 reading frame ends 125 nucleotides beyond the start of the enu coding sequence at uaa51ss-5190, which is labeled t ~ 1 in the figure. were a few 40 s subunits to recognize the nonstandard upstream codons as initiation sites, the resulting 80 s ribosomes-translating in theframe-would bypass the normal enu start site. a dashed line traces the pathway of this shunt. the main point is that ribosomes that terminate at t-1 could reinitiate a t an internal site which would be inaccessible were it not for the shunt. assay the ability to complement a replication-defective (env-) strain of rous sarcoma virus. their results are provocative. point mutations in positions -4 and -7 (i.e., 4 and 7 nucleotides upstream from the aug codon that initiates enu) caused a 10-fold reduction in complementation. on the other hand, the translational efficiency of deletion mutants varied from 5 to 106% of the wild type level, and the variation did not correlate with the presence or absence of any particular portion of the leader sequence. to explain this puzzling pattern (or rather the absence thereof), skalka suggests that the mutations perturb some aspect of secondary structure that is critical for translation. because that idea is difficult to formulate in a way that can be tested, it can do no harm to consider an alternative explanation. the biological assay that was used has the advantage of being exquisitely sensitive, but it has the disadvantage of measuring the yield of enu protein only indirectly: the authors did not show a 10-fold reduction in enu synthesis; they showed a 10-fold drop in complementation. what if complementation were to require, in addition to enu, a second minor protein-either a truncated form of enu that initiates a little farther downstream or a small protein encoded in an alternate reading frame? (there is an open reading frame beginning at aug,,,,-,,,,, for example, that could direct the synthesis of a 10-kda protein.) because the context at the major enu start site is highly favorable, all of 40 s ribosomal subunits that reach that site should initiate there; production of the putative internally initiated protein would therefore require a mechanism for shunting some ribosomes beyond the major enu start site. the hypothesis illustrated in fig. 2 is that a small fraction of the ribosomes initiate within the leader sequence at weak sites (nonstandard codons that lie in a favorable context for initiation) in the -1 reading frame, and translate in that frame past the major enu start site, terminating at the site labeled tin fig. 2 . the small fraction of ribosomes that follow this shunt could reinitiate to produce the second protein postulated above. the notion that the enu gene encodes two products is certainly ad hoc, but it rationalizes the behavior of skalka's mutants. the deleterious mutation in position -4 creates a terminator codon in the -1 reading frame, which would short-circuit the shunt and prevent synthesis of the internally initiated protein. in all of the deletion mutants that fail to complement effkiently, the weak upstream start sites are either in-frame with the major enu start site or terminate upstream from it-again abolishing the shunt. on the other hand, all of the deletion mutants that retain the ability to complement efficiently retain one or more weak upstream start sites (such as gug in position 132-134 in mutant 1371349, or uug in position 22-24 in mutant 65/349) which can feed ribosomes into the shunt. the hypoth-viral translation 269 esis could be tested in two ways. one is to directly measure the yield of the major enu protein-which we predict will not vary, because it is the internally initiated protein that is deficient in these mutants. the best test would make use of a null mutant called pd99/394 that lacks the major enu start site: that mutant should still make the second protein encoded within the enu gene, and therefore should complement all of the other mutants that have lost the shunt. viruses that replicate in the cytoplasm have the potential for coupling transcription with translation. for example, if ribosomes were to bind the 5' end of reovirus mrnas as the nascent chains emerge from the subviral particle, the mrnas would be recruited for translation before the chains grew long enough to fold. that might enhance translation considerably, because the pattern of cleavage by t, rnase suggests that the capped terminus might be sequestered in mature reovirus mrnas (kozak and shatkin, 1978b) . it would be fun to know whether reovirus mrnas are translated more efficiently in naturally infected cells than in cells transfected with cloned viral genes which are transcribed from a plasmid vector. the idea of coupling is ad hoc for reovirus, but there is a glimmer of evidence in the case of silkworm cytoplasmic polyhedrosis virus; whereas performed viral mrnas were inactive in reticulocyte or wheat germ translation systems, viral proteins were synthesized during coupled transcription-translation in frog oocytes (ikegami et al., 1985) . payne and mertens (1983) obtained somewhat different results, in that some viral proteins were made in vitro in the absence of transcription; but the polyhedron protein that predominates in vivo was still not produced in vitro. in the vaccinia virus system, cooper and moss (1978) observed more efficient synthesis of vaccinia proteins when transcription and translation were coupled. synergism could also occur in the opposite direction; i.e., viral transcription might be facilitated by translation. during the early hours of reovirus infection in l cells, transcription is mainly from genome segments m3, s3, s4, and one of the l segments (nonoyama et al., 1974) . because mrnas from segments m3, s3, and s4 bind ribosomes very efficiently (shatkin and kozak, 19831 , one wonders whether preferential transcription is the consequence of preferential tran~1ation.l~ the 17 the hypothesis is complicated, but not necessarily contradicted, by the finding that m3, 53, and s4 are preferentially transcribed in viuo even in the presence of cycloheximide (shatkin and kozak, 1983) . although cycloheximide blocks elongation by 80 s ribosomes, 40 s ribosomal subunits could still bind to the nascent transcripts. possibility that coupled translation enhances transcription was fleetingly entertained for some other cytoplasmic viruses (ball and white, 1978; cooper and moss, 1978) , but the reticulocyte lysate appeared to enhance transcription only because it conferred protection against nucleolytic degradation . it remains possible that transcription and translation are obligatorily coupled in some less well studied rna viruses, as has been hinted for bunyaviruses (patterson and kolakofsky, 1984; pattnaik and abraham, 1983) . in the case of viruses that replicate in the nucleus, the possibility that movement of mrnas out of the nucleus might be coupled with translation has been raised from time to time. coupling clearly is not obligatory, because viral mrnas accumulate in the cytoplasm under many circumstances in which translation is blocked. a good example is the cytoplasmic accumulation of late adenovirus mrnas in the absence of va-rna,. on the other hand, the transport and translation of mrnas are sometimes coordinated. a striking example occurs in adenovirus-infected hela cells that are superinfected with influenza virus : whereas adenovirus blocks both the transport and translation of host mrnas, influenza virus mrnas escape both blocks. the probable mechanism that enables influenza virus mrnas to be translated was discussed in section ii1,e. what mechanism enables influenza mrnas to bypass the block that retains host mrnas in the nucleus? suggested one possibility, namely, an influenza virus-specific transport system. but it seems simpler to look for a single explanation that would account for both the preferential transport and translation. there could be competition at the level of transport, and the same features that make a message highly translatable might make it highly transportable. an alternative view is that the two processes are coupled. one might envision 40 s ribosomal subunits monitoring the nuclear pores, such that only mrnas that can be translated under given circumstances will be transported. along those lines, babiss et az. (1985) have noted that, whereas host mrnas are neither transported nor translated in wildtype adenovirus-infected cells, transport and translation of host mrnas are coordinately restored by mutations in early viral genes that reduce the cytoplasmic accumulation of late viral mrnas. as an extension of the idea that a message will be transported only if it can be translated, one might suggest that mrnas are transported as soon as they become translatable. the consequence would be that translation could sometimes regulate the extent of splicing. some splicing events that could occur, were the transcript kept longer in the nucleus, would be prevented by "prematurely" pulling the mrna out. svensson et al. (1983) invoked this notion to explain some of their observations on the processing of adenovirus early mrnas. coupling of splicing with transport, and transport with translation, would explain why few if any incompletely processed transcripts enter the cytoplasm: no matter how many introns are present in a primary transcript, it remains in the nucleus until every intron has been removed-in effect, until it becomes translatable. it would seem as if the easiest way to judge whether a transcript is translatable is to attempt to translate it. the shutoff of host protein synthesis by herpes simplex virus might not involve a modification in the translational machinery per se. late (second-stage) shutoff is clearly caused by the massive degradation of host mrna. the puzzle of how the nuclease is targeted, such that it degrades host but not viral mrnas, has not yet been solved. a partial explanation might be that herpes virus mrnas are more highly structured, by virtue of their high g + c content. the unusual sensitivity of herpes virus mrnas to hypertonic stress is consistent with the hypothesis that they have extensive secondary structure. the irreversible (read and frenkel, 1983 ) early shutoff of host translation by a structural component of the herpes virion also seems to involve cleavage of host mrnas-enough to inactivate them for translation (fenwick and mcmenamin, 19841 , although they can still be detected by hybridization, more or less (nishioka and silverstein, 1978b; schek and bachenheimer, 1985) . since mutants that are defective in stageone shutoff can still induce secondary shutoff of host protein synthesis (read and frenkel, 19831 , two distinct viral gene products, either nucleases or activators thereof, are apparently involved. a herpes virus mutant that is defective in stage-one host shutoff is defective in switching off the translation of early viral mrnas as well (read and frenkel, 1983) . the differential accumulation of adenovirus early mrnas is also mediated, in part, by the regulated degradation of some transcripts (wilson and darnell, 1981) . degradation of host mrnas might be part of the mechanism by which vaccinia and influenza viruses reduce host translation (see table i ), although clear-cut genetic evidence, such as that described for herpes virus, is lacking in those systems. the extent to which gene expression is regulated by posttranslational proteolytic degradation is probably not fully appreciated. there are striking, isolated examples, for example, the selective degradation of measles virus m protein (sheppard et al., 19851 , the rapid turnover of some early adenovirus proteins (spindler and berk, 1984b) , and stabilization of the cellular protein p53 by its interaction with sv40 t antigen (oren et al., 1981) . given the intricacies of the ubiquitin pathway for proteolysis, it might be surprising were that pathway not perturbed by virus infection. some animal viruses might encode a function that protects foreign (i.e., viral) proteins from degradation, analogous to the pin function of bacteriophage t4 (simon et al., 1983) . although the pattern of codon usage in viral genes is sometimes different from that of the cellular genome, imbalances in the trna pool probably do not affect the yield of most viral proteins because the rate-limiting step is usually initiation rather than elongation. moreover, while there is convincing evidence for a preferred pattern of codon usage in highly expressed bacterial and yeast genes (bennetzen and hall, 1982; ikemura, 1981 ikemura, , 1982 , codon preference seems to be more relaxed in higher eukaryotes (tso et al., 19851 , and therefore the cellular trna pool might not be markedly skewed. consistent with the idea that codon usage is not a major regulatory factor in viral gene expression, the close conservation of amino acid sequences between some viruses is not always accompanied by conservation in the choice of codons (ou et al., 1982) . the degree to which expression might be limited by trna deficiencies has been tested in escherichia coli by using cloned genes that are rich in rare codons. the availability of trna was found to limit translation only when the mrna concentration was extraordinarily high (pedersen, 1984; robinson et al., 1984) . codon usage might regulate translation in more subtle ways, however. one possibility with some experimental justification is that ribosomes pause briefly at rare codons (lizardi et al., 1979; misra and reeves, 1985; varenne et al., 1984) . la discontinuous elongation is not incompatible with efficient translation, as pausing has been detected during the synthesis of some very abundant proteins (cepko and sharp, 1982; lizardi et al., 1979) . slowing translation in certain positions might facilitate folding of the polypeptide and/or its interaction with other components, however. the pattern of codon usage in the signal peptide portion of some genes encourages this notion (spieth et al., 1985) . the suppression of nonsense codons and the occurrence of frameshifting (see section ii,c) might also be facilitated by an imbalance between the cellular trna pool and the viral pattern of codon usage. 1* an alternative explanation for discontinuous elongation is that ribosomes pause when they encounter hairpin structures in the mrna, but that idea is without experimental support. what we have learned about the structure and function of animal virus mrnas can often be extrapolated to cellular mrnas. the mechanism of selecting the initiation site for protein synthesis certainly appears to follow a single formula. the translational machinery displays a certain flexibility (leaky scanning, frameshifting, etc.) that is exploited more frequently by viral than by cellular mrnas. that no (doubt reflects the limited coding capacity of most viral genomes. in contrast, it would seem easier and more efficient for the expansive cellular genome to separately encode two versions of a protein than to attempt to skirt the "monocistronic rule" in the ways described for viruses.lg it is important to remember that there are rules for breaking the monocistronic rule. using those principles, we can correctly predict the qualitative aspects of viral protein synthesis, with very few exceptions. we understand much less about the quantitative aspects of translation, however. although some of the parameters that determine efficiency have been identified in the preceding pages, or at least surmised, we usually cannot predict how efficiently a given mrna will be translated by summing the known parameters. future studies will almost certainly uncover other features that affect translational efficiency: "repressor" proteins, perhaps, or helix-unwinding proteins, or effects of 3'-noncoding sequences, or aspects of mrna primary and secondary structure that are not yet obvious. the suggestion that it is easier to block translation than to enhance it merits repetition. the most efficient mrnas might be those that cannot interact with regulatory rnas, proteins, etc. it is sometimes but not always true that viral mrnas are translated more efficiently than cellular mrnas. i persist in believing that many viruses inhibit cellular protein synthesis inadvertently, and gain little thereby. understanding the mechanism of host shutoff is nonetheless interesting. it might aid in designing virus vectors, and in our understanding of the conditions that promote persistent virus infections (ahmed and fields, 1982) . acknowledgments i thank many colleagues who kindly sent reprints and preprints, and especially those who gave me permission to cite their unpublished observations. in several instances the the 5'-terminal structure of the methylated mrna synthesized in vitro by vsv cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mrnas in extracts from uninfected and infected ascites tumor cells two forms of sv40 tantigen in abortive and lytic infection role of the s4 gene in the establishment of persistent reovirus infection in l cells reversion by hypotonic medium of the shutoff of protein synthesis induced by emc virus translation of capped viral mrnas in poliovirus-infected hela cells protein synthesis in hela cells double-infected with emc virus and poliovirus translation of capped virus mrna in emc virus-infected cells can acg serve as an initiation codon for protein synthesis in eukaryotic cells? sequences at both termini of the 10 genes of reovirus serotype 3 (dearing) the effect of infection with sindbis virus and its temperature sensitive mutants on cellular protein and dna synthesis sequencing studies of pichinde arenavirus s rna indicate a novel coding strategy, an ambisense viral s rna effect of adenovirus on metabolism of specific host mrnas: transport control and specific translational discrimination adenovirus type 5 early region l b gene product is required for efficient shutoff of host protein synthesis adenovirus e1b proteins are required for accumulation of late viral mrna and for effects on cellular mrna translation and transport polyriboadenylic acid preferentially inhibits in vitro translation of cellular compared to vaccinia virus mrnas inhibition of protein synthesis by vaccinia virus coupled transcription and translation in mammalian and avian cell-free systems the replication of picornaviruses expression from an internal aug codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector the number of ribosomes on sv40 late 16s mrna is determined in part by the nucleotide sequence of its leader complete nucleotide sequence of alfalfa mosaic virus rna 3 sequence analysis of hepatitis a virus cdna coding for capsid proteins and rna polymerase direct mapping of adeno-associated virus capsid proteins b and c: a possible acg initiation codon structure of the fmdv translation initiation site and of the structural proteins uag readthrough during tmv rna translation: isolation and sequence of two trnastyr with suppressor activity from tobacco plants the molecular basis for differential translation of tmv rna in tobacco and wheat germ measles virus p gene codes for two proteins inhibition of hela cell protein synthesis during adenovirus infection regulatory mutants of polyoma virus defective in dna replication and the synthesis of early proteins solubilization of a protein synthesis inhibitor from vaccinia virions codon selection in yeast translational interference a t overlapping reading frames in prokaryotic mrna effect of the tripartite leader on synthesis of a nonviral protein in an adenovirus 5 recombinant poliovirus mutant that does not selectively inhibit host cell protein synthesis two small rnas encoded by epstein-barr virus can functionally substitute for the virus-associated rnas in the lytic growth of adenovirus 5 construction and analysis of additional adenovirus substitution mutants confirm the complementation of vai rna function by two small rnas encoded by epstein-barr virus structural requirements of adenovirus vai rna for its translation enhancement function differential inhibition of host cell rna synthesis in several picornavirus-infected cell lines effect of viral infection on host protein synthesis and mrna association with the cytoplasmic cytoskeletal structure intermolecular duplexes formed from polyadenylated vaccinia virus rna the 2.2 kb e1b mrna of human ad12 and ad5 codes for two tumor antigens starting at different aug triplets regulation of protein synthesis in hep2 cells and their cytoplasmic extracts after poliovirus infection globin mrnas are primers for the transcription of influenza viral rna in uitro both the 7-methyl and the 2'-o-methyl groups in the cap of mrna strongly influence its ability to act as primer for influenza virus rna transcription a transcript from the s segment of the germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein sequencing of coronavirus ibv genomic rna: three open reading frames in the 5' "unique" region of mrna d translation of poliovirus rna in uitro: changes in cleavage pattern and initiation sites by ribosomal salt wash initiation factor preparations from poliovirusinfected cells restrict translation in reticulocyte lysates the white pock mutants of rabbit poxvirus: in uitro translation of early host range mutant mrna synthesis of alphavirus-specified rna complex regulation of sv40 earlyregion transcription from different overlapping promoters three intergenic regions of coronavirus mouse hepatitis virus genome rna contain a sequence that is homologous to the 3'-end of the viral mrna leader sequence molecular cloning and complete sequence determination of rna genome of human rhinovirus type 14 permeabilization of cells during animal virus infection sodium ions and the shut-off of host cell protein synthesis by picornaviruses sequences of the s1 genes of the three serotypes of reovirus effects of adenovirus infection on rrna synthesis and maturation in hela cells sequence analysis of the viral core protein and membrane proteins of the flavivirus west nile virus primary structure of the west nile flavivirus genome region coding for all nonstructural proteins effect of poliovirus double-stranded rna on viral and host-cell protein synthesis translation of poliovirus rna in uitro: detection of two different initiation sites regulation of protein synthesis in vsvinfected cells by decreased initiation factor 2 activity assembly of adenovirus major capsid protein is mediated by a nonvirion protein differential translation in normal and adenovirus type 5-infected cells and cell-free systems two initiation sites for foot and mouth disease virus polyprotein in vivo synthesis and processing of sindbis virus nonstructural proteins in uitro overlapping of the vp2-vp3 gene and the vp1 gene in the sv4q genome transcription of vaccinia virus mrna coupled to translation in uitro in uitro translation of immediate early, early, and late classes of rna from vaccinia virus-infected cells discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2 identification of a unique guanine-7-methyltransferase in semliki forest virus infected cell extracts mechanism of interferon action: differential effect of interferon on the synthesis of sv40 and reovirus polypeptides in monkey kidney cells structure-function relationship of rous sarcoma virus leader rna a ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair nucleotide sequence of a viral rna fragment that binds to eukaryotic ribosomes sequence of the 3'-untranslated region of brome mosaic virus coat protein messenger rna genome expression of plant positive-strand rna viruses inhibition of mrna binding to ribosomes by localized activation of dsrna-dependent protein kinase in vitro synthesis of the nonstructural c protein of sendai virus translational specificity in reovirus-infected mouse fibroblasts gene organization of the transforming region of adenovirus type 7 dna initiation of translation of the cauliflower mosaic virus genome from a polycistronic mrna: evidence from deletion mutagenesis oligonucleotide directed mutagenesis of cauliflower mosaic virus dna using a repair-resistant nucleoside analogue identifies an agnogene initiation codon in uitro translation of poliovirus rna: utilization of internal initiation sites in reticulocyte lysate peptide maps and nterminal sequences of polypeptides from early region 1a of human adenovirus 5 catalytic utilization of eif-2 and mrna binding proteins are limiting in lysates from vsv infected l cells host range restriction of vaccinia virus in cho cells: relationship to shutoff of protein synthesis regulation of initiation factors during translational repression caused by serum depletion cellular levels and covalent modification of the subunits of the cap binding protein complex, eif-4f protein synthesis factors 4a and 4b are not altered by poliovirus infection of hela cells complete nucleotide sequence of bacteriophage t7 dna and the locations of t7 genetic elements functional characterization of eukaryotic mrna cap binding protein complex: effects on translation of capped and naturally uncapped rnas sequence relationship of glycosylated and unglycosylated gag polyproteins of moloney murine leukemia virus mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant reovirus hemagglutinin mrna codes for two polypeptides in overlapping reading frames the complete sequence of the m rna of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein analyses of the mrna transcription processes of snowshoe hare bunyavirus s and m rna species human rhinovirus 14 infection of hela cells results in the proteolytic cleavage of the p220 cap binding subunit viral translation proteolysis of a 220,000 dalton polypeptide associated with eif3 and a cap binding protein complex early virion-associated suppression of cellular protein synthesis by herpes simplex virus is accompanied by inactivation of mrna suppression of the synthesis of cellular macromolecules by herpes simplex virus structural difference between the 5' termini of viral and cellular mrna in poliovirus-infected cells: possible basis for the inhibition of host protein synthesis effect of adenovirus infection on expression of human histone genes nucleotide sequence and genome organization of foot and mouth disease virus evidence against the role of k + in the shutoff of protein synthesis by vsv temporal regulation of baculovirus rna: overlapping early and late transcripts early and late functions in a bipartite rna virus: evidence for translational control by competition between viral mrnas bunyavirus nucleoprotein, n, and a nonstructural protein, nss, are coded by overlapping reading frames in the s rna reovirus messenger rna contains a methylated, blocked 5'-terminal structure: m7g(5')ppp(s')gmpcp mechanism of formation of reovirus mrna 5'-terminal blocked and methylated sequence, m7gpppgmpc 5'-terminal structure and mrna stability enhanced poliovirus replication in cytomegalovirus-infected human fibroblasts na+ and k + concentrations and the regulation of the interferon system in chick cells na+ and k + concentrations and the regulation of protein synthesis in sindbis virus-infected chick cells 5'-conformation of capped alfalfa mosaic virus rna 4 may reflect its independence of cap structure or of cap-binding protein for efficient translation sv40 early mrnas contain multiple 5'-termini upstream and downstream from a hogness-goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large t antigen heterogeneity and 5'4erminal structures of the late rnas of sv40 protein synthesis in lysates of aedes albopictus cells infected with vsv sendai virus contains overlapping genes expressed from a single mrna translational discrimination between the four rnas of alfalfa mosaic virus cap accessibility correlates with the initiation efficiency of amv rnas competition between cellular and viral mrnas in uitro is regulated by a messenger discriminatory initiation factor protein synthesis in cells infected with semliki forest virus is not controlled by intracellular cation changes new initiation factor activity required for globin mrna translation inhibition of dna-dependent transcription by the leader rna of vsv sv40 recombinant molecules express the gene encoding p21 transforming protein of harvey murine sarcoma virus sequence of the black beetle virus subgenomic rna and its location in the viral genome nucleotide sequence and genome organization of carnation mottle virus rna utilization of internal aug codons for initiation of protein synthesis directed by mrnas from normal and mutant genes encoding herpes simplex virusspecified thymidine kinase synthesis in vitro of a seven amino acid peptide encoded in the leader rna of rous sarcoma virus sodium and potassium transport in herpes simplex virus-infected cells adenovirus early region 4 encodes functions required for efficient dna replication, late gene expression, and host cell shutoff effects of deletions on expression of the hsv thymidine kinase gene from the intact viral genome: the amino terminus is dispensable for catalytic activity influenza virus mrnas are incomplete transcripts of the genome rnas attenuation of late sv40 mrna synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems large surface proteins of hepatitis v virus containing the pre-s sequence inhibition of host cell protein synthesis by uvinactivated poliovirus control of protein synthesis in extracts from poliovirus-infected cells isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1 virion component of hsv type 1 kos interferes with early shutoff of host protein synthesis induced by hsv type 2 immunological detection of the mrna cap-binding protein site-specific recombination of bacteriophage a: dna sequence of regulatory regions and overlapping structural genes for int and xis characterization of the infections of permissive cells by host range mutants of vsv defective in rna methylation control of expression of the vaccinia virus thymidine kinase gene mapping and identification of the vaccinia virus thymidine kinase gene mutation of a termination codon affects src initiation alfalfa mosaic virus temperature sensitive mutants transcriptioncoupled translation of silkworm cytoplasmic polyhedrosis virus genomic rna in xenopus oocytes correlation between the abundance ofe. coli transfer rnas and the occurrence of the respective codons in its protein genes correlation between the abundance of yeast transfer rnas and the occurrence of the respective codons in protein genes inhibition of host protein synthesis and degradation of cellular mrnas during infection by influenza and hsv constitutive and conditional suppression of exogenous and endogenous genes by anti-sense rna expression of the b u s sarcoma virus pol gene by ribosomal frameshifting biosynthesis of reovirus-specified polypeptides: the reovirus sl mrna encodes two primary translation products biosynthesis of reovirus-specified polypeptides polypeptide cleavages in the formation of poliovirus proteins requirement for adenovirus dna-binding protein and va-i rna for production of adeno-associated virus polypeptides adeno-associated virus proteins: origin of the capsid components identification of the sv40 agnogene product: a dna binding protein further studies on the inhibition of cellular protein synthesis by vsv inhibition of host translation in emc virus-infected l cells: a novel mechanism comparison of initiation rates of emc virus and host protein synthesis in infected cells shutoff of hela cell protein synthesis by emc virus and poliovirus: a comparative study two initiation sites for translation of poliovirus rna in uitro: comparison of lsc and mahoney strains evidence for translational regulation of hsv type 1 gd expression restriction of vsv in a nonpermissive rabbit cell line is at the level of protein synthesis inhibition of cell functions by rna-virus infections lysis gene expression of rna phage ms2 depends on a frameshift during translation of the overlapping coat protein gene deletions of n-terminal sequences of polyoma virus t-antigens reduce but do not abolish transformation of rat fibroblasts role of the avian retrovirus mrna leader in expression: evidence for novel translation control metabolism and expression of rna polymerase i1 transcripts in influenza virus-infected cells nuclear-cytoplasmic transport and vai rna-independent translation of influenza viral mrnas in late adenovirusinfected cells translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eif-2 and selective translation of influenza viral mrnas identification of the components necessary for adenovirus translational control and their utilization in cdna expression vectors viral protein synthesis in barley protoplasts inoculated with native and fractionated brome mosaic virus rna primary structure, gene organization and polypeptide expression of poliovirus rna interferon regulates c-myc gene expression in daudi cells at the post-transcriptional level protein synthesis directed by the rna from a plant virus in a normal animal cell how do eucaryotic ribosomes select initiation regions in messenger rna? inability of circular mrna to attach to eukaryotic ribosomes migration of 40s ribosomal subunits on mrna when initiation is perturbed by lowering magnesium or adding drugs evaluation of the "scanning model" for initiation of protein synthesis in eucaryotes possible role of flanking nucleotides in recognition of the aug initiator codon by eukaryotic ribosomes mechanism of mrna recognition by eukaryotic ribosomes during intiation of protein synthesis analysis of ribosome binding sites from the sl message of reovirus: initiation a t the first and second aug codons comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles translation of insulin-related polypeptides from mrnas with tandemly reiterated copies of the ribosome binding site compilation and analysis of sequences upstream from the translational start site in eukaryotic mrnas selection of initiation sites by eucaryotic ribosomes: effect of inserting aug triplets upstream from the coding sequence for preproinsulin point mutations define a sequence flanking the aug initiator codon that modulates translation by eucaryotic ribosomes influences of mrna secondary structure on initiation by eucaryotic ribosomes migration of 40s ribosomal subunits on mrna in the presence of edeine identification of features in 5'-terminal fragments from reovirus mrna which are important for ribosome binding priming of influenza viral rna transcription by capped heterologous rnas relationship between membrane integrity and the inhibition of host translation in virus-infected mammalian cells characterization of leader 127, 359-366. virus-infected cells rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus synthesis of hepatitis b surface antigen in mammalian cells: expression of the entire gene and the coding region translation of adenovirus serotype 2 late mrnas influenza virus structural and nonstructural proteins in infected cells and their plasma membranes inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus isolation and structural characterization of cap-binding proteins from poliovirus-infected hela cells poliovirus protease 3c (p3-7c) does not cleave p220 of the eucaryotic mrna cap-binding protein complex expression of vaccinia virus early mrnas in ehrlich ascites tumor cells. part of the polysomes at an early stage of virus infection are not bound to the cytoskeleton translation of cellular and viral early mrna in cell-free systems from uninfected and (vaccinia) virusinfected cells a t the early stage mrna discrimination in extracts from uninfected and reovirus-infected l-cells polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit biochemistry of interferons and their actions the cytoskeletal framework and poliovirus metabolism molecular cloning and partial sequencing of hepatitis a viral cdna initiation of translation at internal aug codons in mammalian cells discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells poliovirus protease does not mediate cleavage of the 220,000-da component of the cap binding protein complex translational control of protein synthesis after infection by vsv vsv mrna and inhibition of translation of cellular mrna-is there a p function in vsv? adenovirus tripartite leader sequence enhances translation of mrnas late after infection eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5'-end of mrna the nonstructural proteins of sindbis virus as studied with an antibody specific for the c terminus of the nonstructural readthrough polyprotein differential inhibition of host protein synthesis in l cells infected with rna-temperature-sensitive mutants of vsv arrangment of late rnas transcribed from a 7.1-kb ecori vaccinia virus dna fragment construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus frameshift and intragenic suppressor mutations in a rous sarcoma provirus suggest src encodes two proteins suppression of a vp1 mutant of sv40 by missense mutations in serine codons of the viral agnogene virus-associated rnas of naturally occurring strains and variants of group c adenoviruses nucleotide sequence of the coat protein cistron and the 3'-noncoding region of cucumber green mottle mosaic virus rna influence of the host cell on proteins synthesized by different strains of influenza virus intermediates in the synthesis of tolc protein include an incomplete peptide stalled at a rare arg codon eukaryotic protein synthesis restricted translation of the genome of the flavivirus kunjin in vitro the conformation of adenovirus vai-rna in solution the host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eif2 shutoff of host translation by emc virus infection does not involve cleavage of the eif4f polypeptide that accompanies poliovirus infection inhibition of hela cell protein synthesis by the vaccinia virion irreversible effects of cycloheximide during the early period of vaccinia virus replication formation of the guanylylated and methylated 5'-terminus of vaccinia virus mrna biosynthesis of reovirus-specified polypeptides. multiplication rate but not yield of reovirus serotypes 1 and 3 correlates with the level of virus-mediated inhibition of cellular protein synthesis the regulation of translation in reovirus-infected cells modification of membrane permeability during semliki forest virus infection cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by rauscher murine leukemia virus 35s rna guanidine-sensitive na+ accumulation by poliovirus-infected hela cells adenovirus gene expression: control at multiple steps of mrna biogenesis the complete sequence of the chicken 61 crystallin gene and its 5' flanking region alterations in the protein synthetic apparatus of friend erythroleukemia cells infected with vsv or herpes simplex virus requirement of protein synthesis for the degradation of host mrna in friend erythroleukemia cells infected with hsv type 1 unusual features of the leader sequence of rous sarcoma virus packaging mutant tk15 the 5'-end of poliovirus mrna is not capped with m'g(5')ppp(s') complete nucleotide sequence of the attenuated poliovirus sabin 1 strain genome control of transcription of the reovirus genome vaccinia virusinduced changes in "a+] and [ k + ] in hela cells selective blockage of initiation of host protein synthesis in rna-virus-infected cells a mechanism for the control of protein synthesis by adenovirus va rnal on the regulation of protein synthesis in vaccinia virus infected cells a joint product of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase post-translational regulation of the 54k cellular tumor antigen in normal and transformed cells the influence of the host cell on the inhibition of virus protein synthesis in cells doubly infected with vsv and mengovirus sequence studies of several alphavirus genomic rnas in the region containing the start of the subgenomic rna characterization of a ts mutant of vaccinia virus 25,422-426. novel function that prevents virus-induced breakdown of rna hepatitis b virus genes and their expression in e. coli characterization of lacrosse virus smallgenome transcripts multiple leader rnas and mrnas are transcribed from the lacrosse virus small genome segment identification of four complementary rna species in akabane virus-infected cells the reoviridae methylmercury hydroxide enhancement of translation and transcription of ovalbumin and conalbumin mrnas e . coli ribosomes translate in viuo with variable rate leaky uag termination codon in tobacco mosaic virus rna characteristics of a coupled cellfree transcription and translation system directed by vaccinia cores insertion mutagenesis to increase secondary structure within the 5'moncoding region of a eukaryotic mrna reduces translational efiiciency evidence for the presence of an inhibitor on ribosomes in mouse l cells infected with mengovirus regulation of herpesvirus macromolecular synthesis. properties of a polypeptides made in hsv-1 and hsv-2 infected cells utilization of messenger in adenovirus-2-infected cells at normal and elevated temperatures a frameshift mutation in the pre-s region of the human hepatitis b virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin characterization of ribosome binding on rous sarcoma virus rna in uitro translation of mulv and msv rnas in nuclease-treated reticulocyte extracts: enhancement of the gag-pol polypeptide with yeast suppressor trna proteins specified by avian erythroblastosis virus: coding region localization and identification of a previously undetected erb-b polypeptide molecular cloning of poliovirus cdna and determination of the complete nucleotide sequence of the viral genome cell-free translation of frog virus 3 mrnas herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of q (immediate early) viral polypeptides the genome of simian virus 40 regulation of a protein synthesis initiation factor by adenovirus va-rnai interferon-mediated, doubled-stranded rna-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells vaccinia virus induces cellular mrna degradation double-stranded rnadependent protein kinase and 2-5a system are both activated in interferon-treated, emc virus-infected hela cells nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution structure of the flavivirus genome 5' and 3' terminal nucleotide sequences of the rna genome segments of influenza virus codon usage can affect efficiency of translation of genes in e. coli inhibition of translation by poliovirus: inactivation of a specific initiation factor 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facilitates the initiation of translation in virus-infected cells rna prevents phosphorylation of eif2a subsequent to infection nucleotide sequence of rous sarcoma virus interferon, double-stranded rna, and protein phosphorylation reovirus inhibition of cellular rna and protein synthesis: role of the s4 gene the reoviridae capping of eucaryotic mrnas molecular mechanisms of virus-mediated cytopathology mrna cap binding proteins: essential factors for initiating translation the reoviridae complete nucleotide sequence of the neuraminidase gene of influenza b virus a previously unrecognized b virus glycoprotein from a bicistronic mrna that also encodes the viral neuraminidase measles virus matrix protein synthesized in a subacute sclerosing panencephalitis cell line translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo nucleotide sequence of moloney murine leukaemia virus mechanism of translational control by partial phosphorylation of the a subunit of eukaryotic initiation factor 2 translational control by adenovirus: lack of va-rna, during adenovirus infection results in phosphorylation of eif-2 and inhibition of protein synthesis alterations in the protein synthetic apparatus of cells infected with herpes simplex virus stabilization of proteins by a bacteriophage t4 gene cloned in e . coli coding sequence of coronavirus mhv-jhm mrna 4 coronavirus mhv-jhm mrna 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame reovirus-induced modification of cap-dependent translation in infected l cells regulation of translation in l-cells infected with reovirus cytoplasmic methionine transfer rnas from eukaryotes extraction and fingerprint of sv40 large and small t-antigens production of human beta interferon in insect cells infected with a baculovirus expression vector differential stimulation of capped mrna translation in uitro by cap binding protein probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins coronavirus mrna synthesis involves fusion of non-contiguous sequences translation efficiency of zein mrna is reduced by hybrid formation between the 5'-and 3'-untranslated region the nucleotide sequence of a nematode vitellogenin gene translation efficiency of adenovirus early region 1a mrnas deleted in the 5' untranslated region rapid intracellular turnover of adenovirus 5 early region 1a proteins herpes simplex virus-induced changes in cellular and adenovirus rna metabolism in an adenovirus type 5-transformed human cell line the effect of hypertonic conditions on protein synthesis in cells infected with herpes virus sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon adenovirus va rnai: a positive regulator of mrna translation adenovirus va rnai mediates a translational stimulation which is not restricted to the viral mrnas splicing of adenovirus 2 early region 1a mrnas is non-sequential a cell-free model of the emc virus-induced inhibition of host cell protein synthesis two forms of purified m7g-cap binding protein with different effects on capped mrna translation in extracts of uninfected and poliovirus-infected hela cells the location of v-src in a retrovirus vector determines whether the virus is toxic or transforming adenovirus vai rna is required for efficient translation of viral mrnas a t late times after infection the hepatitis b virus complete nucleotide sequences of all three poliovirus serotype genomes isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cdnas: genomic complexity and molecular evolution of the gene shutoff of neuroblastoma cell protein synthesis by semliki forest virus: loss of ability of crude initiation factors to recognize early sfv and host mrnas infection of neuroblastoma cells by sfv translation is a nonuniform process. effects of trna availability on the rate of elongation of nascent polypeptide chains translational control of early protein synthesis at the late stage of vaccinia virus infection mutational alterations within the sv40 leader segment generate altered 16s and 19s mrnas individual hsv transcripts: characterization of specific genes. jn "the herpesviruses nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1 the role of mrna competition in regulating translation a single uga codon functions as a natural termination signal in the coliphage q p coat protein cistron disruption of the three cytoskeletal networks 199-203 marilyn kozak in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock protein synthesis in bhk-21 cells infected with semliki forest virus characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase nucleotide sequence of an immediateearly frog virus 3 gene macromolecular synthesis in cells infected by frog virus 3 further genetic localization of the transforming sequences of the p21 v-ras gene of harvey murine sarcoma virus control of mrna concentration by differential cytoplasmic half-life evidence that a g u a u e a and ccaagmga initiate translation in the same mrna in region e3 of adenovirus differential phosphorylation of soluble versus ribosome-bound eif2 in the ehrlich ascites tumor cell resistance to inhibitors of mammalian cell protein synthesis induced by preincubation in hypertonic growth medium murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease splicing in adenovirus and other animal viruses s. cereuisiae ribosomes recognize non-aug initiation codons studies on the intracellular synthesis of reovirus-specified proteins 