Wild-type Vesicular Stomatitis Virus Research Articles

Overproduction of Double-Stranded RNA in Vesicular Stomatitis Virus-Infected Cells Activates a Constitutive Cell-Type-Specific Antiviral Response

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.

Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

Recombinant vesicular stomatitis virus vectors as oncolytic agents in the treatment of high-grade gliomas in an organotypic brain tissue slice-glioma coculture model.

The purpose of this study was to evaluate both replication-competent and replication-restricted recombinant vesicular stomatitis virus (VSV) vectors as therapeutic agents for high-grade gliomas by using an organotypic brain tissue slice-glioma coculture system. The coculture system involved growing different brain structures together to allow neurons from these tissues to develop synaptic connections similar to those found in vivo. Rat C6 or human U87 glioma cells were then introduced into the culture to evaluate VSV as an oncolytic therapy. The authors found that recombinant wild-type VSV (rVSV-wt) rapidly eliminated C6 glioma cells from the coculture, but also caused significant damage to neurons, as measured by a loss of microtubule-associated protein 2 immunoreactivity and a failure in electrophysiological responses from neurons in the tissue slice. Nonetheless, pretreatment with interferon beta (IFNbeta) virtually eliminated VSV infection in healthy tissues without impeding any oncolytic effects on tumor cells. Despite the protective effects of the IFNbeta pretreatment, the tissue slices still showed signs of cytopathology when exposed to rVSV-wt. In contrast, pretreatment with IFNbeta and inoculation with a replication-restricted vector with its glycoprotein gene deleted (rVSV-deltaG) effectively destroyed rat C6 and human U87 glioma cells in the coculture, without causing detectable damage to the neuronal integrity and electrophysiological properties of the healthy tissue in the culture. Data in this study provide in vitro proof-of-principle that rVSV-deltaG is an effective oncolytic agent that has minimal toxic side effects to neurons compared with rVSV-wt and therefore should be considered for development as an adjuvant to surgery in the treatment of glioma.

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity.

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.

Cutting edge: inflammatory signals drive organ-specific autoimmunity to normally cross-tolerizing endogenous antigen.

Links have been observed between infections and the development of autoimmunity. Proposed explanations include activation of self-Ag-bearing APC. Using a model system in which transgenic OVA is expressed in enterocytes, we showed that CD8 T cell recognition of cross-presented Ag in gut-associated lymph nodes was tolerogenic. However, concomitant infection with vesicular stomatitis virus encoding OVA abrogated tolerance and induced disease. We now show that following transfer of naive OT-I T cells, the addition of wild-type vesicular stomatitis virus, oral cholera toxin, or CD40 triggering can induce intestinal disease in transgenic mice. Tissue damage accompanied dramatic increases in cytokine release by activated OT-I cells in the intestine. The data indicated that products of antigenically unrelated infections can combine with cross-presented self-Ags on APC to prime autoaggressiveness, independent of additional Ag release. These results help explain how diverse pathogens, lacking any homology to self-proteins, could be causative agents in induction of organ-specific autoimmunity.

Vesicular Stomatitis Virus Glycoprotein Containing the Entire Green Fluorescent Protein on Its Cytoplasmic Domain Is Incorporated Efficiently into Virus Particles

The envelope glycoprotein (G) of vesicular stomatitis virus (VSV) contains a short cytoplasmic domain of 29 amino acids. To determine whether VSV particle assembly could accommodate a G protein with a large cytoplasmic domain, we constructed a gene called G/GFP encoding the VSV G protein with the 27-kDa green fluorescent protein linked to its cytoplasmic domain. This gene was inserted into the infectious clone of VSV and we recovered a recombinant virus expressing G/GFP from this extra gene. This VSV-G/GFP virus grew to titers equivalent to that of wild-type virus and was stable upon passaging. The G/GFP protein formed mixed trimers containing an average of two wild-type G proteins and one G/GFP protein. This heterotrimeric protein was expressed on the cell surface, and was incorporated into virus particles with almost the same efficiency as wild-type VSV G protein. These results indicate that there is substantial space available between the viral membrane and the nucleocapsid that can accommodate such a large cytoplasmic domain. The green fluorescent virus particles were readily visualized by fluorescence microscopy and had a normal morphology by electron microscopy. To determine whether virus assembly could occur efficiently when all G proteins contained the GFP cytoplasmic domain, a VSV recombinant in which the G gene was completely replaced by the VSV-G/GFP gene was recovered. This virus rapidly lost expression of the GFP protein sequence through introduction of a stop codon within the sequence encoding the G cytoplasmic domain, indicating strong selection against homotrimeric G protein bearing such a large cytoplasmic domain.

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release.

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.

Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III.

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)- RNA synthesis were measured by incorporation of [3H]uridine. v6 and tsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)- RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and alpha-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.

Vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge.

Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982-5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.

Chimeric measles viruses with a foreign envelope.

Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.

Attenuation of Recombinant Vesicular Stomatitis Viruses Encoding Mutant Glycoproteins Demonstrate a Critical Role for Maintaining a High pH Threshold for Membrane Fusion in Viral Fitness

A plasmid-based recovery system was used to generate four unique vesicular stomatitis virus (VSV) mutants that encode glycoproteins (G proteins) with single or double amino acid substitutions in two conserved acidic residues adjacent to the putative G protein fusion domain. Previously we demonstrated that three of the mutant G proteins (D137-L, E139-L, and DE-SS) have slightly reduced pH thresholds for membrane fusion activity. In this report we show that even though the viruses encoding D137-L, E139-L, and DE-SS were recovered with high efficiency, these mutants were attenuated for growth in cell culture. Plaque formation was significantly delayed with these mutants and the plaques were smaller and more diffuse than those produced by wild-type VSV. In addition, cells infected with these mutants produced approximately 5- to 10-fold less infectious virus than cells infected with a similarly recovered VSV encoding the wild-type G protein. Using R18-labeled virus we found that the mutant G proteins had approximately 50% of the fusion activity of wild-type G at pH 6.3 and only 75% activity at pH 5.8. We also show that the mutant viruses were more sensitive to chloroquine inhibition of infection than either wild-type VSV or the mutant E139-T, which has a fusion phenotype similar to wild-type G protein. Reduced fusion activity and attenuation of infectivity was not due to differences in the amount of G protein incorporated into virions, nor to differences in the amount of virus binding to cells at physiological pH. Although infectivity was assayed at neutral pH, we observed an increase in virus binding with both mutant and wild-type virions as the pH was lowered, and the increase in binding occurred near the pH threshold for membrane fusion activity. From these data we propose a model in which VSV entry involves an increase in virus binding to the inner leaflet of the endosomal membrane during endosome acidification. Concomitant with this higher affinity binding, G protein becomes primed to initiate fusion of the viral envelope with the endosomal membrane. Viruses with mutations that delay the onset of increased binding and fusion lag behind wild-type VSV in their ability to initiate a productive infection, potentially because the location within the cytoplasm where these viruses ultimately fuse is not optimal for either virus uncoating or replication of the viral genome.

Reversible and irreversible steps in assembly and disassembly of vesicular stomatitis virus: equilibria and kinetics of dissociation of nucleocapsid-M protein complexes assembled in vivo.

The matrix (M) protein of vesicular stomatitis virus (VSV) condenses the viral nucleoprotein core (nucleocapsid) into a tightly coiled, helical nucleocapsid-M protein (NCM) complex. Using NCM complexes assembled in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein binding to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in their M proteins were analyzed using sedimentation and light-scattering assays. At physiological ionic strength, the binding reaction had the characteristics of a dynamic reversible equilibrium. A temperature-sensitive M protein mutant lost the ability of M protein to reversibly dissociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast to the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaCl) were not able to bind M protein at low ionic strength with the same high affinity seen in NCM complexes assembled in vivo. The effect of incubation at 250 mM NaCl was shown to be due to a change in nucleocapsids rather than a change in soluble M protein. This result supports the idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.

Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells That Affects Inhibition of Host-Directed Gene Expression

In addition to its function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression in cells persistently infected with VSV. Viruses isolated from L cells persistently infected with VSV inhibited host RNA synthesis more slowly than wild-type (wt) VSV. M genes of the persistent viral population were cloned and sequenced. One mutation, an N to D change at position 163 of the protein sequence (N163D), was common to all the molecular clones. The N163D M protein was synthesized from transfected mRNA at a rate that was 30% of that of wt M protein, but was turned over at a rate that was similar to that of wt M protein. Transfection of mRNA encoding N163D M protein inhibited expression of a cotransfected target gene encoding chloramphenicol acetyl transferase (CAT), but the inhibition was 6 to 10 times less effective than transfection of equivalent amounts of wt M mRNA. This difference could not be accounted for by differences in translation of CAT mRNA. Thus, when the differences in M protein expression were taken into account, N163D M protein was 2 to 3 times less effective than wt M protein in the inhibition of host-directed gene expression, similar to the differences in host transcription observed in virus-infected cells. Point mutations in addition to the N163D mutation were found in about half of the M gene molecular clones. The M gene of an independently isolated molecular clone, N163D.2, contained two additional point mutations in its carboxy terminal region. N163D.2 M protein was highly defective in inhibition of host gene expression and was turned over more rapidly than wt M protein. These results support the idea that M gene mutations contribute to a reduced cytopathic effect in cells persistently infected with VSV.

The vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter.

In cells infected by wild-type (wt) vesicular stomatitis virus (VSV) Indiana, host transcription is severely inhibited. DNA cotransfection studies have implicated the VSV matrix (M) protein in this process (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). The M protein inhibited transcription not only from viral promoters in plasmids but also from the chromosomally integrated human immunodeficiency virus type 1 (HIV-1) provirus promoter (S.-Y. Paik, A. C. Banerjea, G. G. Harmison, C.-J. Chen, and M. Schubert, J. Virol. 69:3529-3537, 1995). In this study, we investigated the effect of wt VSV M protein on expression of a reporter gene under control of a cellular promoter (beta-interferon [IFN-beta] promoter), using double transient transfections in BHK and COS-1 cells. The cellular IFN-beta promoter was as susceptible to the inhibitory effect of the M protein as the viral promoters used previously. Viral proteins N, P, and G had no significant effect on reporter gene expression. The M protein gene from VSV mutant T1026R1, which is defective in host transcription inhibition, was cloned and sequenced, and its effect on reporter gene expression was tested. The mutant M protein had a methionine-to-arginine change at position 51 in the protein sequence and did not inhibit transcription from either the IFN-beta promoter or viral promoters. This VSV mutant is a good inducer of IFN, as opposed to the wt virus, which suppresses IFN induction. These results show that the M protein inhibits transcription from cellular as well as viral promoters and that the M protein does not regulate the IFN promoter any differently from viral promoters. While the M protein may play a role in IFN gene regulation, other viral or cellular factors that provide specificity to the induction process must also be involved.

Large-population passages of vesicular stomatitis virus in interferon-treated cells select variants of only limited resistance.

Vesicular stomatitis virus (VSV) populations were repeatedly passaged in L-929 cells treated with alpha interferon (IFN-alpha) at levels of 25 U/ml. This IFN-alpha concentration induced a 99.9% inhibition of viral yield in standard infections. Analysis of viral fitness (overall replicative ability measured in direct competition with a reference wild-type VSV) after 21 passages in IFN-treated cells showed only a limited increase or no increase in fitness, compared with the greater increase upon parallel passage in cells not treated with IFN-alpha. However, this limited increase in fitness was more pronounced when competition assays were carried out with IFN-alpha-treated cells, suggesting the selection of VSV populations with a low level of resistance to IFN-alpha. Thus, despite the extensively documented capacity of VSV to adapt to changing environments, the antiviral state induced by IFN-alpha imposes adaptive constraints on VSV which are not readily overcome.

Glycan-dependent and -independent Association of Vesicular Stomatitis Virus G Protein with Calnexin

Calnexin (CNX) is a membrane-bound molecular chaperone that associates with newly synthesized proteins in the endoplasmic reticulum. Although several studies have indicated that it interacts exclusively with glycoproteins that carry monoglucosylated N-linked oligosaccharides, others have reported that it can bind to proteins that have no glycans. To address this discrepancy, we translated wild-type vesicular stomatitis virus G protein and nonglycosylated mutant forms in the presence of microsomes and examined their association with CNX. Individual G protein molecules were found to efficiently associate with CNX when both glycans were present and less efficiently if there was only a single glycan. Nonglycosylated G protein also interacted with CNX, but only when misfolded and present in high molecular weight aggregates. The results indicated that CNX can interact with G protein in two ways: through an oligosaccharide-dependent mechanism that involves individual substrate proteins; and in an oligosaccharide-independent association with large aggregates.

NF-κB Activation Is Delayed in Mouse L929 Cells Infected with Interferon Suppressing, but Not Inducing, Vesicular Stomatitis Virus Strains

Vesicular stomatitis virus (VSV) mutant T1026R1 of the Indiana (IN) serotype is a good inducer of interferon (IFN). This mutant was used to study the activation of NF-κB, a transcription factor necessary for IFN induction, in mouse L929 cells that were stably transfected with a chimeric gene containing the human IFN-β gene promoter attached to the chloramphenicol acetyltransferase (CAT) coding sequence. NF-κB DNA binding activity was detected as early as 30 min after virus adsorption in nuclear extracts, increased up to 4 hr, and then remained constant for at least 6 additional hr. The kinetics of CAT expression correlated with the kinetics of NF-κB nuclear DNA binding activity. Virus entry and delivery of viral components into the cytoplasm were required for NF-κB activation. Exposure of T1026R1 to one hit of UV irradiation nearly completely reduced NF-κB activation. In cells infected with wild-type (wt) VSV (IN), a noninducer of IFN, NF-κB DNA binding activity in the nucleus was delayed for several hours after virus adsorption. Coinfection of wt VSV and T1026R1 resulted in the reduction of T1026R1-promoted NF-κB activation. This inhibitory activity of wt VSV was abolished by one hit of UV irradiation. Under similar conditions expression of the CAT gene was more UV resistant, suggesting that IFN gene expression is regulated at multiple levels.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.