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Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

ABSTRACT: Hepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2 alpha and attenuates ISG protein expression despite normal ISG mRNA induction. ISG protein induction is restored and the antiviral effects of interferon are enhanced when PKR expression is suppressed in interferon-treated infected cells. Whereas host protein translation, including antiviral ISGs, is suppressed by activated PKR, HCV IRES-dependent translation is not. These results suggest that the ability of HCV to activate PKR may, paradoxically, be advantageous for the virus during an IFN response by preferentially suppressing the translation of ISGs.

SUBMITTER: Garaigorta U

PROVIDER: S-EPMC2905238 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

Garaigorta Urtzi U Chisari Francis V FV

Cell host & microbe 20091201 6

Hepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2 al ...[more]

PMID: 20006840

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Project description:The negative regulators in the interferon (IFN) signaling pathway inhibit intrahepatic immune response, resulting in suboptimal therapeutic response to IFNα treatment in chronic hepatitis B (CHB) patients. Identifying the key negative factors and elucidating the regulating mechanism are essential for improving anti-HBV (hepatitis B virus) efficacy of IFNα. From the Gene Expression Omnibus (GEO) database, we downloaded and analyzed gene expression profiles of CHB patients with different responses to IFNα (GSE54747), and found that innate immune status was associated with the IFNα-based therapeutic response in CHB patients. Through PCR array, we found higher baseline level of IFN-induced transmembrane protein 2 (IFITM2) mRNA and lower baseline level of IFNα mRNA in peripheral blood mononuclear cells (PBMCs) of CHB patients with suboptimal response to IFNα treatment. Increased IFITM2 protein was also found in the serum of IFNα nonresponsive patients. With further experiments, we found that overexpressing IFITM2 in Huh7 cells suppressed endogenous IFNα synthesis by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3); knocking out IFITM2 enhanced activation of the endogenous IFNα synthesis pathway, exhibiting better inhibition on HBV replication. We also found that IFITM2 protein was shuttled by exosomes to dendritic cells (DCs), the main source of endogenous IFNα. Exosome-mediated transport of IFITM2 inhibited synthesis of endogenous IFNα in DCs whereas the inhibitory effect was abolished when IFITM2 was knocked out. Furthermore, we demonstrated that both palmitoylation inhibitor and mutation on 70/71 sites of IFITM2 protein influenced its incorporation into exosomes. Mutated IFITM2 protein increased the effect of IFNα against HBV. Conclusion: Exosome-mediated transport of IFITM2 to DCs inhibits IFNα pathway activation and blocks anti-HBV efficacy of exogenous IFNα. The findings provide an explanation to the suboptimal response of CHB patients to IFNα treatment.

Project description:Background & aimsHepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach.MethodsWe performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further.ResultsThe screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, β 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, β isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFNα; these genes represent a heretofore unknown class of non-IFN-stimulated gene IEGs.ConclusionsWe performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFNα against human pathogenic viruses. We found that IFNα restricts HCV via actions of general and specific IEGs.

Project description:UnlabelledHepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.ImportanceWhile the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

Project description:UnlabelledHepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-α) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B.ImportanceOur study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

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Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

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Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

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