scholarly journals MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus

2016 ◽  
Vol 90 (14) ◽  
pp. 6598-6610 ◽  
Author(s):  
Ghizlane Maarifi ◽  
Zara Hannoun ◽  
Marie Claude Geoffroy ◽  
Faten El Asmi ◽  
Karima Zarrouk ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Mrna Synthesis ◽  
Restriction Factors ◽  
Viral Protein Synthesis ◽  
Independent Manner ◽  
Content Type ◽  
Intracellular Pool ◽  
Vesicular Stomatitis ◽  
Cellular Pathways ◽  
Opposing Effects

ABSTRACTMultiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of theRhabdoviridaefamily, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction.IMPORTANCEWe report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

scholarly journals Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis

2008 ◽  
Vol 83 (4) ◽  
pp. 1930-1940 ◽  
Author(s):  
Jianrong Li ◽  
Amal Rahmeh ◽  
Vesna Brusic ◽  
Sean P. J. Whelan
Keyword(s):  
Amino Acid ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Full Length ◽  
Single Amino Acid ◽  
Mrna Synthesis ◽  
L Protein ◽  
Vesicular Stomatitis ◽  
Opposing Effects ◽  
Gene Junction

ABSTRACT The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3′-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3′ termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5′ cap formation and 3′ polyadenylation.

scholarly journals Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Louis-Marie Bloyet ◽  
Benjamin Morin ◽  
Vesna Brusic ◽  
Erica Gardner ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Binding Domain ◽  
Genome Replication ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Vesicular Stomatitis ◽  
Oligomerization Domain

ABSTRACT Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

scholarly journals Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
James Duehr ◽  
Teddy John Wohlbold ◽  
Lisa Oestereich ◽  
Veronika Chromikova ◽  
Fatima Amanat ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vesicular Stomatitis Virus ◽  
Viral Vector ◽  
Virus Disease ◽  
Protective Efficacy ◽  
Influenza Viruses ◽  
Content Type ◽  
Zaire Ebolavirus ◽  
Vesicular Stomatitis

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 −/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

Orobol: An Inhibitor of Vesicular Stomatitis Virus that Blocks the Synthesis of Viral Nucleic Acids and the Glycosylation of G Protein

1994 ◽  
Vol 5 (2) ◽  
pp. 99-104 ◽  
Author(s):  
M. J. Almela ◽  
A. Irurzun ◽  
L. Carrasco
Keyword(s):  
Protein Synthesis ◽  
Nucleic Acids ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Viral Protein Synthesis ◽  
Viral Translation ◽  
Naturally Occurring ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Virus Influenza

The naturally occurring isoflavonoid orobol exhibits antiviral effects against some animal viruses. Addition of the compound after virus entry inhibits the appearance of late viral protein synthesis in Vesicular Stomatitis Virus, influenza, or vaccinia virus-infected cells, but has no effect on poliovirus protein synthesis. Concentrations of the compound above 10–50 Mg ml−1 are sufficient to decrease the synthesis of VSV proteins when added early during infection, but have no effect on viral translation if added later, indicating that orobol does not block VSV translation directly. The synthesis of VSV nucleic acids is one of the targets of this flavonoid. The synthesis of both minus and plus-stranded viral RNA are inhibited by orobol when added during the first 2 h of infection. In addition, this compound interferes potently with the glycosylation of VSV G protein, indicating that orobol has several targets of antiviral action. The possibility that orobol interferes with the function of the cellular vesicular system is discussed.

scholarly journals A Monomeric GTPase-Negative MxA Mutant with Antiviral Activity

2000 ◽  
Vol 74 (17) ◽  
pp. 8202-8206 ◽  
Author(s):  
Christian Janzen ◽  
Georg Kochs ◽  
Otto Haller
Keyword(s):  
Antiviral Activity ◽  
Vesicular Stomatitis Virus ◽  
Vero Cells ◽  
Virus Multiplication ◽  
Gtpase Activity ◽  
Wild Type ◽  
Gtp Hydrolysis ◽  
Intracellular Pool ◽  
Antiviral Function ◽  
Vesicular Stomatitis

ABSTRACT MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.

scholarly journals Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Farooq Nasar ◽  
Demetrius Matassov ◽  
Robert L. Seymour ◽  
Theresa Latham ◽  
Rodion V. Gorchakov ◽  
...  
Keyword(s):  
Single Dose ◽  
Vesicular Stomatitis Virus ◽  
Clinical Studies ◽  
Vaccine Vector ◽  
Vaccine Vectors ◽  
Content Type ◽  
Multivalent Vaccine ◽  
Equine Encephalitis Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

ABSTRACT The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans. IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

scholarly journals Polymerase Slippage at Vesicular Stomatitis Virus Gene Junctions To Generate Poly(A) Is Regulated by the Upstream 3′-AUAC-5′ Tetranucleotide: Implications for the Mechanism of Transcription Termination

2001 ◽  
Vol 75 (15) ◽  
pp. 6901-6913 ◽  
Author(s):  
John N. Barr ◽  
Gail W. Wertz
Keyword(s):  
Nucleotide Sequence ◽  
Vesicular Stomatitis Virus ◽  
Transcription Termination ◽  
Nucleotide Composition ◽  
Minimum Length ◽  
Mrna Synthesis ◽  
Reiterative Transcription ◽  
Vesicular Stomatitis ◽  
Polymerase Slippage ◽  
Termination Process

ABSTRACT Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3′-AUAC-5′, the U7 tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3′ poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U7tract. Previously we determined that in addition to the AUAC tetranucleotide, the U7 tract was an essential signal in the termination process. Shortening or interrupting the U7tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U7 tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U7 tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U7 tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis.

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