Inhibition of Host and Viral Translation during Vesicular Stomatitis Virus Infection

Connor, John H.; Lyles, Douglas S.

doi:10.1074/jbc.m501156200

Cited by 71 publications

(41 citation statements)

References 31 publications

(18 reference statements)

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“…On the other hand, our findings that eIF2␣ is phosphorylated and that VSV protein synthesis is arrested in DTT-treated cells indicate both that VSV does not express factors for preventing PERK-mediated eIF2 phosphorylation and that translation of VSV mRNAs is very sensitive to phosphorylation of this initiation factor, as has been previously documented (34). Taken together, these results suggest that ISGs other that PKR are involved in the IFN-induced arrest of VSV replication in avian cells, a situation already reported for the replication of this virus in mammalian cells (73)(74)(75)(76).…”

Section: Discussionsupporting

confidence: 69%

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

Lostalé‐Seijo

Martı́nez-Costas

Benavente

2016

J Virol

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We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells. IMPORTANCE Type I interferons constitute the first barrier of defense against viral infections, and one of the best characterized antiviral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV)by PKR-dependent and -independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV toward IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins. I nterferons (IFNs) comprise a family of multifunctional cytokines that were originally discovered by their strong antiviral activity and which are now recognized as the first barrier that viruses must overcome to establish a productive infection. Of the three IFN types, type I interferon (IFN-␣/␤) displays the highest antiviral activity, and its expression is induced in many cell types by viral infection or following c...

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Section: Discussionsupporting

confidence: 69%

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

Lostalé‐Seijo

Martı́nez-Costas

Benavente

2016

J Virol

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“…Other functions have been ascribed to the orthologous region of VSV M, including regulation of membrane association (66) and protein turnover (59), although both of those studies utilized larger deletions or multiple amino acid mutations than the point mutation used here. Outside this specific coil domain, the N terminus of VSV M has been implicated in other aspects of host inhibition, including association with RAE1/NUP98 (45,48) or suppression of eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation (M51R) (67). Whether VHSV M also engages these conserved proteins and/or whether D62A mutations impact these same cellular functions, directly or indirectly, will have to await future studies.…”

Section: Discussionmentioning

confidence: 99%

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

Qin

Weaver

Pore

et al. 2017

J Virol

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Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/ luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virusinduced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

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“…Much of the decrease in protein synthesis is caused by phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) 4 at Ser-51 by eIF2␣ kinases that respond specifically to stress (1)(2)(3)(4). Heme-regulated eIF2␣ kinase or heme-regulated inhibitor (HRI) is a member of the eIF2␣ kinase family that controls globin synthesis in response to the heme concentration in reticulocytes (5)(6)(7)(8).…”

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confidence: 99%

Elucidation of the Heme Binding Site of Heme-regulated Eukaryotic Initiation Factor 2α Kinase and the Role of the Regulatory Motif in Heme Sensing by Spectroscopic and Catalytic Studies of Mutant Proteins

Igarashi

Murase

Iizuka

et al. 2008

Journal of Biological Chemistry

141

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Heme-regulated eukaryotic initiation factor 2␣ (eIF2␣) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2␣, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory CysPro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.Eukaryotic cells decrease their overall rates of protein synthesis for survival in response to a variety of stress conditions, such as shortage of amino acids, UV light illumination, virus infection, and accumulation of denatured proteins. Much of the decrease in protein synthesis is caused by phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) 4 at Ser-51 by eIF2␣ kinases that respond specifically to stress (1-4). Heme-regulated eIF2␣ kinase or heme-regulated inhibitor (HRI) is a member of the eIF2␣ kinase family that controls globin synthesis in response to the heme concentration in reticulocytes (5-8). HRI is inactive at normal heme concentrations. Under conditions of heme deficiency, the enzyme is activated by autophosphorylation and subsequently phosphorylates eIF2␣ at Ser-51. In addition to globin, HRI controls the synthesis of tryptophan 2,3-dioxygenase and cytochrome P450 2B in liver upon acute porphyria (9, 10). Thus, HRI is possibly one of the most important existing heme sensor protein families for eukaryote survival in response to cell emergency states.Heme-responsive/sensing proteins, also known as "heme sensor proteins" are a current focus of investigation. In these proteins heme association (or dissociation) per se regulates various important physiological functions, such as transcription, proteolysis, and kinase activity (11...

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Inhibition of Host and Viral Translation during Vesicular Stomatitis Virus Infection

Cited by 71 publications

References 31 publications

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

Elucidation of the Heme Binding Site of Heme-regulated Eukaryotic Initiation Factor 2α Kinase and the Role of the Regulatory Motif in Heme Sensing by Spectroscopic and Catalytic Studies of Mutant Proteins

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