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Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells., (© 2020 Wiley Periodicals LLC.)

The phosphoprotein (P) of the nonsegmented negative-sense RNA viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (P MD ), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, P MD , for viral gene expression and virus growth. A truncated P protein lacking the central dimerization domain (P ΔMD ) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type virus, showing that the dimerization domain of P is dispensable for viral gene expression and virus replication in cell culture. Because RNA viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new viruses. IMPORTANCE The phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA viruses, including some major human pathogens (e.g., rabies virus, measles virus, respiratory syncytial virus [RSV], Ebola virus, and Nipah virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different viruses and, in some cases, hijacks cell components to the advantage of the virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA viruses., (Copyright © 2020 American Society for Microbiology.)

Viral vectors derived from vesicular stomatitis virus (VSV) are important vectors for the development of vaccines and for the treatment of cancer. The efficiency of therapy based on VSV is dependent on the dose of virus used. Therefore it is essential to measure accurately and reproducibly the amount of functional vectors in the samples to be tested. Two common methods used to measure the titer of VSV are TCID 50% and plaque assay. In the current study, we compared these two titration methods by using a recombinant VSV expressing the green fluorescent protein (VSV-GFP) as a model virus. Some culture media developed for suspension mammalian cells contain dextran sulfate. We observed that plaque assay, but not TCID 50% , can underestimate the virus titer up to 10 fold when VSV-GFP was produced in culture media containing dextran sulfate. Dextran sulfate is commonly used in serum-free culture media to reduce cell aggregation in suspension culture. The inhibitory effect of dextran sulfate on the titration of VSV-GFP was confirmed by supplementing the culture medium with this compound during virus production. Our results also demonstrated that extending the incubation time during plaque assay and TCID 50% increases virus titer., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Developments in genetic engineering have allowed researchers and clinicians to begin harnessing viruses to target and kill cancer cells, either through direct lysis or through recruitment of antiviral immune responses. Two powerful viruses in the fight against cancer are the single-stranded RNA viruses vesicular stomatitis virus and Zika virus. Here, we describe methods to propagate and titer these two viruses. We also describe a simple cell-killing assay to begin testing modified viruses for increased potential killing of glioblastoma cells.

During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif-Glu169, Phe171, and Leu174-to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid. IMPORTANCE During viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis., (Copyright © 2018 American Society for Microbiology.)

The matrix protein of vesicular stomatitis virus (VSV) performs multiple functions during viral genome replication and virion production and is involved in modulating multiple host signaling pathways that favor virus replication. To perform numerous functions within infected cells, the M protein needs to recruit cellular partners. To better understand the role of M during VSV replication, we looked for interacting partners by using the two-hybrid system. The eukaryotic translation initiation factor 3, subunit i (eIF3i) was identified to be an M-binding partner, and this interaction was validated by GST pull-down and laser confocal assays. Through a mutagenesis analysis, we found that some mutants of M between amino acids 122 and 181 impaired but did not completely abolish the M-eIF3i interaction. Furthermore, the knockdown of eIF3i by RNA interference decreased viral replication and transcription in the early stages but led to increase in later stages. VSV transcription was increased at 4h post-infection but was not changed at 8 and 12h post-infection after the over-expression of eIF3i. Finally, we also demonstrated that VSV could inhibit the activity of Akt1 and that the knockdown of eIF3i inhibited the expression of the ISGs regulated by phospho-Akt1. These results indicated that eIF3i may affect VSV growth by regulating the host antiviral response in HeLa cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis.

Unlabelled: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high case fatality rate caused by SFTS virus (SFTSV). Effective vaccines and specific therapies for SFTS are urgently sought, and investigation into virus-host cell interactions is expected to contribute to the development of antiviral strategies. In this study, we have developed a pseudotype vesicular stomatitis virus (VSV) bearing the unmodified Gn/Gc glycoproteins (GPs) of SFTSV (SFTSVpv). We have analyzed the host cell entry of this pseudotype virus and native SFTSV. Both SFTSVpv and SFTSV exhibited high infectivity in various mammalian cell lines. The use of lysosomotropic agents indicated that virus entry occurred via pH-dependent endocytosis. SFTSVpv and SFTSV infectivity was neutralized by serial dilutions of convalescent-phase patient sera. Entry of SFTSVpv and growth of SFTSV were increased in Raji cells expressing not only the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) but also DC-SIGN-related (DC-SIGNR) and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). 25-Hydroxycholesterol (25HC), a soluble oxysterol metabolite, inhibited the cell entry of SFTSVpv and the membrane fusion of SFTSV. These results indicate that pH-dependent endocytosis of SFTSVpv and SFTSV is enhanced by attachment to certain C-type lectins. SFTSVpv is an appropriate model for the investigation of SFTSV-GP-mediated cell entry and virus neutralization at lower biosafety levels. Furthermore, 25HC may represent a potential antiviral agent against SFTS., Importance: SFTSV is a recently discovered bunyavirus associated with SFTS, a viral hemorrhagic fever with a high case fatality rate endemic to China, South Korea, and Japan. Because little is known about the characteristics of the envelope protein and entry mechanisms of SFTSV, further studies will be required for the development of a vaccine or effective therapies. In this study, we investigated the mechanism of SFTSV cell entry using SFTSVpv and the native virus. SFTSV can grow in nonsusceptible cell lines in the presence of certain C-type lectins. Moreover, 25HC, an oxysterol metabolite, may represent a potential therapeutic inhibitor of SFTSV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-β and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-β (boIFN-β; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-β and demonstrate the activation of a general antiviral state after EHMT2 inhibition.

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface., (Copyright © 2015, Drokhlyansky et al.)

We have utilized the enveloped viral model to study the effect of fluvastatin on membrane trafficking in isolated rat myofibers. Our immunofluorescence studies constantly showed that infections in myofibers, which were treated with fluvastatin prior and during the infection with either vesicular stomatitis virus (VSV) or influenza A virus, propagated more slowly than in control myofibers without drug treatment. Experiments with a virus expressing Dad1 tagged with green fluorescent protein (GFP-Dad1) showed that fluvastatin did not affect its distribution within the ER/SR network and immunofluorescence staining for GM130 did not show any marked effect on the structure of the Golgi components. Furthermore, fluvastatin did not inhibit trafficking of the chimeric transport marker VSV temperature sensitive G protein (tsG-GFP) from the ER to the Golgi. We next subjected VSV infected myofibers for pulse-chase labeling experiments and found that fluvastatin did not slow down the ER-to-Golgi trafficking or Golgi to plasma membrane trafficking of the viral glycoprotein. These studies show that fluvastatin inhibited the propagation of viral infection in skeletal myofibers but no adverse effect on the exocytic trafficking could be demonstrated. These results suggest that other effects of statins rather than inhibition of ER-to-Golgi trafficking might be behind the myotoxic effects of the statins., (© 2015 International Federation for Cell Biology.)

This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.

Non-segmented negative strand RNA viruses belonging to the Mononegavirales order possess RNA-dependent RNA polymerase L proteins within viral particles. The L protein is a multifunctional enzyme catalyzing viral RNA synthesis and processing (i.e., mRNA capping, cap methylation, and polyadenylation). Using vesicular stomatitis virus (VSV) as a prototypic model virus, we have shown that the L protein catalyzes the unconventional mRNA capping reaction, which is strikingly different from the eukaryotic reaction. Furthermore, co-transcriptional pre-mRNA capping with the VSV L protein was found to be required for accurate stop?start transcription to synthesize full-length mRNAs in vitro and virus propagation in host cells. This article provides a review of historical and present studies leading to the elucidation of the molecular mechanism of VSV mRNA capping.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Technology for producing biologically active recombinant alpha2b-interferon is based on creating a bacterial producer strain containing the cDNA of human interferon alpha. The authors have obtained two producers of recombinant alpha2b-interferon, the synthesis of the target protein in them occurs in the inclusion bodies. The schemes of isolation and purification of biologically active recombinant alpha2b-interferon have been developed. The drug purity was approximately 97-98%. Biological activity in the culture of sensory cells in the cytopathic test was 4.2*10(8)ME/mg.

SIRT1, the closest mammalian homolog of yeast Sir2, is an NAD(+)-dependent deacetylase with relevant functions in cancer, aging, and metabolism among other processes. SIRT1 has a diffuse nuclear localization but is recruited to the PML nuclear bodies (PML-NBs) after PML upregulation. However, the functions of SIRT1 in the PML-NBs are unknown. In this study we show that primary mouse embryo fibroblasts lacking SIRT1 contain reduced PML protein levels that are increased after reintroduction of SIRT1. In addition, overexpression of SIRT1 in HEK-293 cells increases the amount of PML protein whereas knockdown of SIRT1 reduces the size and number of PML-NBs and the levels of PML protein in HeLa cells. SIRT1 stimulates PML sumoylation in vitro and in vivo in a deacetylase-independent manner. Importantly, the absence of SIRT1 reduces the apoptotic response of vesicular stomatitis virus-infected cells and favors the extent of this PML-sensitive virus replication. These results show a novel function of SIRT1 in the control of PML and PML-NBs.

Since Vero cells are currently considered as an acceptable cell substrate to produce a wide range of viruses, we developed a virus production platform using Vero cells adapted to grow in suspension in serum-free media. After adapting anchorage-dependent Vero cells to grow as a free-cell suspension, vesicular stomatitis virus, herpes simplex virus 1 and polio virus 1 production rates were evaluated in batch cultures using spinner flasks and perfused cultures in a bioreactor. The achieved results constitute valuable information for the development of a low-cost high-productivity process using a suspension culture of Vero cells to produce viral vaccines.

Innate immunity to viruses involves receptors such as Retinoic Acid Induced Gene-1 (RIG-I), which senses viral RNA and triggers a signaling pathway involving the outer mitochondrial membrane protein mitochondrial antiviral signaling (MAVS). Recent work has identified that NLRX1, a member of another class of innate immune receptors, sequesters MAVS away from RIG-I and thereby prevents mitochondrial antiviral immunity. In this study, we demonstrate that the proteasome PSMA7 (alpha4) subunit associates with MAVS in vivo and in vitro. Expression of PSMA7 results in a potent inhibition of RIG-1 and MAVS-mediated IFN-beta promoter activity; conversely, depletion of PSMA7 with small interference RNA enhances virus-induced type I IFN production, with consequent reduction of virus replication. Furthermore, a striking reduction in the abundance of endogenous MAVS with overexpressed PSMA7 was found and virus infection leads to transient increase in the endogenous PSMA7 protein level. Cumulatively, these results suggest that PSMA7 is a negative regulator of the MAVS-mediated innate immunity that probably serves to attenuate the establishment of an antiviral state during viral infection, highlighting the biological significance of PSMA7-MAVS association as an important cellular regulatory control.

Overexpression of myeloid cell leukemia 1 protein (Mcl-1), an anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member, contributes to chemotherapy resistance of tumors. The short half-life of Mcl-1 makes it an interesting target for therapeutic agents that negatively interfere with cellular protein biosynthesis, such as oncolytic viruses. Vesicular Stomatitis Virus (VSV) has been established as the oncolytic virus that efficiently disrupts de novo protein biosynthesis of infected cells. Here, we show that after VSV infection, Mcl-1 protein levels rapidly declined, whereas the expression of other members of the Bcl-2 family remained unchanged. Mcl-1 elimination was a consequence of proteasomal degradation, as overexpression of a degradation-resistant Mcl-1 mutant restored Mcl-1 levels. Mcl-1 rescue inhibited apoptosis and thereby confirmed that Mcl-1 downregulation contributes to VSV-induced apoptosis. In vitro, VSV virotherapy in combination with chemotherapy revealed an enhanced therapeutic effect compared with the single treatments, which could be reverted by Mcl-1 rescue or RNA interference (RNAi)-mediated knockdown of pro-apoptotic Bax and Bak proteins. Finally, in a tumor mouse model, combinations of doxorubicin and VSV showed a superior therapeutic efficacy compared with VSV or doxorubicin alone. In summary, our data indicate that VSV virotherapy is an attractive strategy to overcome tumor resistance against conventional chemotherapy by elimination of Mcl-1.

The plaque assay has long served as the "gold standard" to measure virus infectivity and test antiviral drugs, but the assay is labor-intensive, lacks sensitivity, uses excessive reagents, and is hard to automate. Recent modification of the assay to exploit flow-enhanced virus spread with quantitative imaging has increased its sensitivity. Here we performed flow-enhanced infection assays in microscale channels, employing passive fluid pumping to inoculate cell monolayers with virus and drive infection spread. Our test of an antiviral drug (5-fluorouracil) against vesicular stomatitis virus infections of BHK cell monolayers yielded a two-fold improvement in sensitivity, relative to the standard assay based on plaque counting. The reduction in scale, simplified fluid handling, image-based quantification, and higher assay sensitivity will enable infection measurements for high-throughput drug screening, sero-conversion testing, and patient-specific diagnosis of viral infections.

Defective interfering particles (DIPs) are virus-like particles that arise during virus growth, fail to grow in the absence of virus, and replicate at the expense of virus during co-infections. The inhibitory effects of DIPs on virus growth are well established, but little is known about how DIPs influence their own growth. Here vesicular stomatitis virus (VSV) and its DIPs were used to co-infect BHK cells, and the effect of DIP dose on virus and DIP production was measured using a yield-reduction assay. The resulting dose-response data were used to fit and evaluate mathematical models that employed different assumptions. Our analysis supports a multiple-hit process where DIPs inhibit or promote virus and DIP production, depending on dose. Specifically, three regimes of co-infection were apparent: (i) low DIP - where both virus and DIPs are amplified, (ii) medium DIP - where amplification of both virus and DIPs is inhibited, and (iii) high DIP - with limited recovery of virus production and further inhibition of DIP growth. In addition, serial-passage infections enabled us to estimate the frequency of de novo DIP generation during virus amplification. Our combined experiments and models provide a means to understand better how DIPs quantitatively impact the growth of viruses and the spread of their infections.

Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.

Given the lethality of H5N1 avian influenza viruses (AIV) and the recurring spread from poultry to humans, an effective vaccine against H5N1 viruses may be needed to prevent a pandemic. We generated experimental vaccine vectors based on recombinant vesicular stomatitis virus (VSV) expressing the H5 hemagglutinin (HA) from an H5N1 virus isolated in 1997. The HA gene was expressed either from an attenuated wild-type VSV vector or from a single-cycle vector containing a deletion of the VSV G gene. We found that all of the vectors induced potent neutralizing antibody titers against the homologous and antigenically heterologous H5N1 viruses isolated in 2004 and 2005. Vaccination of mice with any combination of prime or prime/boost vectors provided long-lasting protection (>7 months) against challenge with AIV, even in animals receiving a single dose of single-cycle vaccine. Our data indicate that these recombinants are promising vaccine candidates for pandemic influenza.

Several viruses have been demonstrated to be the etiologic agent in chronic progressive diseases, associated with persistence; however, major questions concerning the pathogenic mechanisms of viral persistence are still unanswered. With the aim of identifying host cellular proteins that may play a role in viral replication, we established long-term persistently infected human glioblastoma cell lines with mutant measles virus (MV) and analyzed the host proteins by two-dimensional gel electrophoresis (2-DE) with mass spectrometry. We observed significant down-modulation in the expression of mitochondrial short chain enoyl-CoA hydratase (ECHS), which catalyzes the beta-oxidation pathway of fatty acid. Knockdown of this gene by a short interference RNA (siRNA) apparently impaired wild-type MV replication and the cytopathic effects (CPEs) of MV were significantly reduced in siRNA-transfected cells. These findings will shed light upon a new important notion for the interaction between virus replication and lipid metabolism in host cells and might provide a new strategy for virus control.

A number of studies have demonstrated that non-neuronal acetylcholine can play a role in the regulation of T cell function. Recently, we reported that CD8(+) T cells, from mice with a targeted deletion of the M(1) muscarinic receptor, had a defect in differentiating into cytolytic T lymphocytes when stimulated in vitro. In the current report, we analyze the in vivo function of CD8(+) T cells from mice with targeted deletions of either M(1) or M(5) muscarinic receptors. M(1) or M(5) knockout mice were infected with either lymphocytic choriomeningitis virus or vesicular stomatitis virus. Expansion of anti-viral CD8(+) T cells was monitored by staining with tetramer reagents specific for the immunodominant peptides of the viruses. No defect in expansion of CD8(+) T cells was observed in either M(1) or M(5) knockout mice. The extent to which one can draw a generalized conclusion that M(1) and M(5) are not involved in anti-viral immunity depends upon issues of antigen strength, genetic background, induction of redundant receptors, and the potential for qualitative defects in the expanded CD8(+) T cells.

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.

The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.

Sinefungin (SIN), a natural S-adenosyl-L-methionine analog produced by Streptomyces griseolus, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2'-O (2'-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2'-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SINR) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G-L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SINR mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SINR viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.

How do the fitness effects of several mutations combine? Despite its simplicity, this question is central to the understanding of multilocus evolution. Epistasis (the interaction between alleles at different loci), especially epistasis for fitness traits such as reproduction and survival, influences evolutionary predictions "almost whenever multilocus genetics matters". Yet very few models have sought to predict epistasis, and none has been empirically tested. Here we show that the distribution of epistasis can be predicted from the distribution of single mutation effects, based on a simple fitness landscape model. We show that this prediction closely matches the empirical measures of epistasis that have been obtained for Escherichia coli and the RNA virus vesicular stomatitis virus. Our results suggest that a simple fitness landscape model may be sufficient to quantitatively capture the complex nature of gene interactions. This model may offer a simple and widely applicable alternative to complex metabolic network models, in particular for making evolutionary predictions.

A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD(50)) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD(50) of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.

Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

In a companion paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:492-502, 2007), we provided indirect evidence that cell-type-specific growth restriction of the vesicular stomatitis virus (VSV) polR mutants may be due to enhanced production of double-stranded RNA (dsRNA). We show here that polR growth in mouse L-929 cells was rescued by vaccinia virus coinfection and that sole expression of the vaccinia virus dsRNA-binding E3L protein, via coinfection with an engineered VSV minigenome, also restored polR growth. Expression of dsRNA-binding protein NS1A or NS1B from influenza virus, but not C protein from Sendai virus, which does not bind dsRNA, likewise effected polR rescue. The N-terminal dsRNA-binding domain of NS1A, only 73 amino acids in length, but not a full-size mutant NS1A lacking dsRNA-binding activity, restored polR growth. Both key aspects of polR growth restriction, namely inhibition of genome replication and release of low-infectivity virus particles, were countered by expression of the dsRNA-binding proteins. We tested the effects of overproducing dsRNA in wild-type VSV infections by coinfecting cells with a VSV recombinant expressing the sense strand of the enhanced green fluorescent protein gene (VSV-GFP) and one expressing the antisense strand (VSV-PFG). These coinfections mimicked all aspects of polR restriction, including host range, lack of effect on transcription, reduced virus particle infectivity, and insensitivity to inhibition of host gene transcription or dsRNA-activated protein kinase activity. We conclude that, for some cell types, overproduction of dsRNA during VSV infection triggers an immediate and constitutive host cell antiviral effector response independent of interferon induction or signaling.

Pharmacologic inhibition of the proteasome resulted in increased NOS-1 protein levels and increased NO production by neuronal cells. This correlated with an increased antiviral effect of IFN-gamma against the replication of vesicular stomatitis virus (VSV) replication in vitro. We also observed that a regulatory protein, Protein Inhibitor of NOS-1 (PIN) was down-regulated by IFN-gamma treatment, and more ubiquitinated PIN accumulated in IFN-gamma treated neurons. In cells of the reticuloendothelial system, IFN-gamma treatment induces the expression of a set of low molecular weight MHC-encoded proteins (LMPs), which replace the beta-subunit of the proteasome complex during the proteasome neosynthesis, resulting in a complex termed the immunoproteasome. LMP2, -7, and -10 were induced and the immunoproteasome was generated by IFN-gamma treatment in neuronal cells. Importantly, we observed that IFN-gamma induced inhibition of VSV protein synthesis was not dependent on ubiquitination.

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (approximately 30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.

Background: Virotherapy represents a novel therapeutic modality for the treatment of malignant diseases. Vesicular stomatitis virus (VSV) has been shown to exert antitumor effect in several tumor types. Since the potential oncolytic activity of VSV has not yet been evaluated in epithelial tumors of the conjunctiva, we set out to investigate the susceptibility of the immortalized Wong-Kilbourne derivative of the Chang conjunctival cell line (WK) to VSV and analyze the role of apoptosis in VSV-mediated induction of cell death., Methods: WK cells were infected with VSV at various multiplicities and maintained for different periods of time. VSV-infected cells were analyzed by inverted microscopy for the development of cytopathic effects (CPE). Virus replication was measured by indirect immunofluorescence assay, Western blot analysis and plaque titration. The apoptotic response of the infected cells was quantitated by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of Bcl-2 and Bax proteins., Results: The WK cell line was highly permissive to VSV replication and was highly susceptible for the CPE of this virus. VSV infection elicited the apoptotic death of WK cells. Mock-infected cells exhibited endogenous expression of Bcl-2 and p21 Bax proteins. VSV infection caused a significant decrease in the expression level of Bcl-2. Moreover, in parallel with a slight decrease in the level of p21 Bax, p18 Bax protein accumulated in VSV-infected WK cells., Conclusions: VSV is a powerful inducer of apoptosis in immortalized WK cells. The VSV-mediated alterations in the expressions of Bcl-2 and Bax proteins may play important roles in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. This virus may possess oncolytic activity in epithelial tumors of the conjunctiva.

The proper selection of size and shape for polyethylene glycol (PEG) is one of the most important points in PEGylation technology. Therefore, PEGs of various sizes and shapes have been widely developed to endow specific properties. In this study, a unique, trimer-structured, 43 kDa PEG was conjugated to interferon-alpha 2a (IFN) by forming an amide bond to improve the pharmacokinetic properties and minimize the loss of IFN bioactivity. Mono-PEGylated IFN (PEG(3)-IFN) prepared by utilizing this unique PEG was purified and characterized by cation-exchange chromatography and MALDI-TOF mass spectrometry. The in vitro bioactivity, in vivo stability, and pharmacokinetics of PEG(3)-IFN were examined and compared to those of native IFN. PEG(3)-IFN exhibited comparable in vitro bioactivities to native IFN and an excellent stability of the conjugation linkage in rat serum and various organs following subcutaneous injection. Furthermore, it showed slow absorption and markedly reduced clearance in rats, thereby increasing the biological half-life by about 40-fold compared to that of native IFN. This is the first report on the application of unique, trimer-structured PEG to bioactive proteins. The results suggest that unique, trimer-structured 43 kDa PEG can provide some advantages to improve the pharmacokinetic properties and to maintain the bioactivity of therapeutic proteins in clinical use.

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.

We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism that is well defined genetically in Caenorhabditis elegans. RNAi has been postulated to function as an adaptive antiviral immune mechanism in the worm, but there is no experimental evidence for this. Part of the limitation is that there are no known natural viral pathogens of C. elegans. Here we describe an infection model in C. elegans using the mammalian pathogen vesicular stomatitis virus (VSV) to study the role of RNAi in antiviral immunity. VSV infection is potentiated in cells derived from RNAi-defective worm mutants (rde-1; rde-4), leading to the production of infectious progeny virus, and is inhibited in mutants with an enhanced RNAi response (rrf-3; eri-1). Because the RNAi response occurs in the absence of exogenously added VSV small interfering RNAs, these results show that RNAi is activated during VSV infection and that RNAi is a genuine antiviral immune defence mechanism in the worm.

A quantitative understanding of the innate immune response will enable its recruitment against emerging, poorly characterized, or weaponized viral pathogens. To gain insights into how the innate responses can limit viral spread, we used quantitative focal infections to study how the spread of recombinant vesicular stomatitis viruses (VSV) on baby hamster kidney (BHK) and delayed brain tumor (DBT) cell monolayers is affected by innate cellular antiviral responses. We observed that rates of infection spread correlated with one-step growth rankings for four ectopic VSV strains: N1, N2, N3, and N4. However, this correlation was lost for M51R, a recombinant VSV mutant that lacks the ability to shut-off host gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1, even though their one-step growth was comparable. In DBT cells, M51R infections failed to spread beyond the site of inoculation. Addition of anti-interferon antibody restored M51R spread and one-step growth to wild-type levels. Interestingly, the antibody enhanced the spread of wild-type virus but not its growth. These results suggest that while the rate of viral spread generally correlates with the rate of viral growth, the induction of cellular antiviral activities can be in some cases, the overriding factor in both spread and growth. In summary, focal infections enabled us to visualize and quantify how viral spread was inhibited by cellular antiviral activities. This study demonstrates a mechanism for quantifying how innate cellular responses can mitigate infection spread in vitro.

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Influenza C virus contains two envelope glycoproteins: CM2, a putative ion channel protein; and HEF, a unique multifunctional protein that performs receptor-binding, receptor-destroying and fusion activities. Here, it is demonstrated that expression of HEF is sufficient to pseudotype replication-incompetent vesicular stomatitis virus (VSV) that lacks the VSV glycoprotein (G) gene. The pseudotyped virus showed characteristic features of influenza C virus with respect to proteolytic activation, receptor usage and cell tropism. Chimeric glycoproteins composed of HEF ectodomain and VSV-G C-terminal domains were efficiently incorporated into VSV particles and showed receptor-binding and receptor-destroying activities but, unlike authentic HEF, did not mediate efficient infection, probably because of impaired fusion activity. HEF-pseudotyped VSV efficiently infected polarized Madin-Darby canine kidney cells via the apical plasma membrane, whereas entry of VSV-G-complemented virus was restricted to the basolateral membrane. These findings suggest that pseudotyping of viral vectors with HEF might be useful for efficient apical gene transfer into polarized epithelial cells and for targeting cells that express 9-O-acetylated sialic acids.

To determine the vector competence of Culicoides sonorensis Wirth & Jones midges for vesicular stomatitis virus (VSV)-New Jersey, insects were experimentally infected per os and sampled over time. Viral replication, as determined by in situ hybridization, was seen in epithelial, neural, and hemolymph cell types throughout the insect. Spatial and temporal distribution of virus was determined by immunohistochemical examination of sequentially sampled insects. Tissues of the alimentary canal were infected in a temporal pattern that paralleled the route of digestion/absorption: foregut and midgut by day 1, surrounding hemolymph and Malpighian tubules by day 3, and finally the midgut/ hindgut junction, hindgut, and rectal region by day 5. The circulation of virus in the hemolymph by day 3 coincided with infection of the dermis and fat bodies, the salivary glands, eyes, cerebral and subthoracic ganglia, and the ovaries. Oviduct epithelium and ovarial sheaths were infected by day 3, followed by infection of the developing oocytes by day 5. Interestingly, neural infections were seen in the subabdominal ganglia innervating the midgut in 33% of insects by 1 d postfeeding in the absence of positive staining in the hemolymph or surrounding tissues. A retrograde axonal transport infection route for these ganglia is discussed. The disseminated, productive, noncytolytic infection in Culicoides is consistent with that of an efficient biological vector for VSV. Virus readily replicated throughout the insect, passing both midgut and salivary gland infection barriers and reaching transmission-related organs in 3 d. Establishing the competence of this insect vector for VSV provides the foundation for animal transmission studies in the future. The possibility of horizontal, transovarial, and mechanical transmission is discussed.

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.

Due to their extremely high genetic diversity, which is a direct consequence of high mutation rates, RNA viruses are often described as molecular quasispecies. According to this theory, RNA virus populations cannot be understood in terms of individual viral clones, as they are clouds of interconnected mutants, but this prediction has not yet been demonstrated experimentally. The goal of this study was to determine the fitness of individual clones sampled from a given RNA virus population, a necessary previous step to test the above prediction. To do so, limiting dilutions of a vesicular stomatitis virus population were employed to isolate single viral clones and their initial growth dynamics were followed, corresponding to the release of the first few hundred viral particles. This technique is useful for estimating basic fitness parameters, such as intracellular growth rate, viral yield per cell, rate at which cells are infected and time spent in cell-to-cell transmission. A combination of these parameters allows estimation of the fitness of individual clones, which seems to be determined mainly by their ability to complete infection cycles more quickly. Interestingly, fitness was systematically higher for initial clones than for their derived populations. In addition to environmental changes, such as cellular defence mechanisms, these differences are attributable to high RNA virus mutation rates.

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.