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Antagonistic effects of cellular poly(C) binding proteins on vesicular stomatitis virus gene expression.

ABSTRACT: Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections.

SUBMITTER: Dinh PX

PROVIDER: S-EPMC3165775 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Antagonistic effects of cellular poly(C) binding proteins on vesicular stomatitis virus gene expression.

Dinh Phat X PX Beura Lalit K LK Panda Debasis D Das Anshuman A Das Anshuman A Pattnaik Asit K AK

Journal of virology 20110713 18

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of ...[more]

PMID: 21752917

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Project description:Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

Project description:BACKGROUND:The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. METHODS:We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. RESULTS:The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. CONCLUSIONS:This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV?G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

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Antagonistic effects of cellular poly(C) binding proteins on vesicular stomatitis virus gene expression.

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Antagonistic effects of cellular poly(C) binding proteins on vesicular stomatitis virus gene expression.

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