Analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach

<p>Abstract</p> <p>Background</p> <p>Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and the best studied member of the order <it>Mononegavirales</it>. There is now compelling evidence that enveloped virions released from infected cells carry numerous host (cellular) proteins some of which may play an important role in viral replication. Although several cellular proteins have been previously shown to be incorporated into VSV virions, no systematic study has been done to reveal the host protein composition for virions of VSV or any other member of <it>Mononegavirales</it>.</p> <p>Results</p> <p>Here we used a proteomics approach to identify cellular proteins within purified VSV virions, thereby creating a "snapshot" of one stage of virus/host interaction that can guide future experiments aimed at understanding molecular mechanisms of virus-cell interactions. Highly purified preparations of VSV virions from three different cell lines of human, mouse and hamster origin were analyzed for the presence of cellular proteins using mass spectrometry. We have successfully confirmed the presence of several previously-identified cellular proteins within VSV virions and identified a number of additional proteins likely to also be present within the virions. In total, sixty-four cellular proteins were identified, of which nine were found in multiple preparations. A combination of immunoblotting and proteinase K protection assay was used to verify the presence of several of these proteins (integrin β1, heat shock protein 90 kDa, heat shock cognate 71 kDa protein, annexin 2, elongation factor 1a) within the virions.</p> <p>Conclusion</p> <p>This is, to our knowledge, the first systematic study of the host protein composition for virions of VSV or any other member of the order <it>Mononegavirales</it>. Future experiments are needed to determine which of the identified proteins have an interaction with VSV and whether these interactions are beneficial, neutral or antiviral with respect to VSV replication. Identification of host proteins-virus interactions beneficial for virus would be particularly exciting as they can provide new ways to combat viral infections via control of host components.</p

An unexpected inhibition of antiviral signaling by virus-encoded tumor suppressor p53 in pancreatic cancer cells

AbstractVirus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro

Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane- associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positivestrand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5\u27-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2

Identification of Sendai Virus L Protein Amino Acid Residues Affecting Viral mRNA Cap Methylation▿

Viruses of the order Mononegavirales all encode a large (L) polymerase protein responsible for the replication and transcription of the viral genome as well as all posttranscriptional modifications of viral mRNAs. The L protein is conserved among all members of the Mononegavirales and has six conserved regions (“domains”). Using vesicular stomatitis virus (VSV) (family Rhabdoviridae) experimental system, we and others recently identified several conserved amino acid residues within L protein domain VI which are required for viral mRNA cap methylation. To verify that these critical amino acid residues have a similar function in other members of the Mononegavirales, we examined the Sendai virus (SeV) (family Paramyxoviridae) L protein by targeting homologous amino acid residues important for cap methylation in VSV which are highly conserved among all members of the Mononegavirales and are believed to constitute the L protein catalytic and S-adenosylmethionine-binding sites. In addition, an SeV L protein mutant with a deletion of the entire domain VI was generated. First, L mutants were tested for their abilities to synthesize viral mRNAs. While the domain VI deletion completely inactivated L, most of the amino acid substitutions had minor effects on mRNA synthesis. Using a reverse genetics approach, these mutations were introduced into the SeV genome, and recombinant infectious SeV mutants with single alanine substitutions at L positions 1782, 1804, 1805, and 1806 or a double substitution at positions 1804 and 1806 were generated. The mutant SeV virions were purified, detergent activated, and analyzed for their abilities to synthesize viral mRNAs methylated at their cap structures. In addition, further studies were done to examine these SeV mutants for a possible host range phenotype, which was previously shown for VSV cap methylation-defective mutants. In agreement with a predicted role of the SeV L protein invariant lysine 1782 as a catalytic residue, the recombinant virus with a single K1782A substitution was completely defective in cap methylation and showed a host range phenotype. In addition, the E1805A mutation within the putative S-adenosylmethionine-binding site of L resulted in a 60% reduction in cap methylation. In contrast to the homologous VSV mutants, other recombinant SeV mutants with amino acid substitutions at this site were neither defective in cap methylation nor host range restricted. The results of this initial study using an SeV experimental system demonstrate similarities as well as differences between the L protein cap methylation domains in different members of the Mononegavirales

Effect of RBV on cell viability of seven cell lines.

<p>To determine the relative toxicity of increasing concentrations of RBV in different cell lines, 80%-confluent uninfected cells were treated with RBV for 24 h and tested for viability using MTT cell viability assay (A) or CellTiter-Glo Luminescent Cell Viability Assay (B) or by cell counting using trypan blue dye exclusion as an indicator of live cells (C) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011265#s2" target="_blank">Materials and Methods</a>. To determine the sensitivity of the MTT assay, serial dilutions of A549 and HeLa cells were plated [lower left and right graphs in (A)], grown for 24 h, cells from separate wells were trypsinized and counted using a hemocytometer (36,000 cells for HeLa and 38,000 cells for A549 formed 100% confluent monolayers), and MTT assay was conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011265#s2" target="_blank">Materials and Methods</a>. (A–C) The data (done in triplicate) represent the mean ± standard deviation and are expressed as a percentage of the untreated control. Statistical analysis was done using one-way ANOVA with Tukey's post hoc test (GraphPad Prism 4, San Diego, CA). ***P<0.001, **P<0.01, *P<0.05, as compared to mock-treated cells (indicated as 0 µM RBV).</p

Cellular Proteins Associated with the Interior and Exterior of Vesicular Stomatitis Virus Virions

<div><p>Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact (“whole”) virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.</p></div

Viral infectivity and replication kinetics in the seven cell lines.

<p>(A) Cells were infected with serial dilutions of VSV-GFP (left) or SeV-GFP (right), and infectious foci were counted to calculate the infectivity of the viral stock for each cell line. (B) One-step kinetics of viral replication in seven cell lines. Cells were infected in parallel with VSV-GFP or SeV-GFP at MOI of 3 CIU/cell (1 h absorption), washed 3 times with PBS, and kept in SFM. The media containing newly generated virions was collected at the indicated time points and viral titrations were performed on BHK21 (for VSV) or Vero cells (for SeV).</p