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Enhancement of interferon induction by ORF3 product of hepatitis E virus.

ABSTRACT: Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-? mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-? promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-? expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion.Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.

SUBMITTER: Nan Y

PROVIDER: S-EPMC4135948 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Enhancement of interferon induction by ORF3 product of hepatitis E virus.

Nan Yuchen Y Ma Zexu Z Wang Rong R Yu Ying Y Kannan Harilakshmi H Fredericksen Brenda B Zhang Yan-Jin YJ

Journal of virology 20140521 15

<h4>Unlabelled</h4>Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level ...[more]

PMID: 24850742

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Project description:UnlabelledAll major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN-γ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-γ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA.ImportanceInterferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

Project description:Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.

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Enhancement of interferon induction by ORF3 product of hepatitis E virus.

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Enhancement of interferon induction by ORF3 product of hepatitis E virus.

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