title: Mechanistic link between the anti-HCV effect of interferon gamma and control of viral replication by a Ras-MAPK signaling cascade.
abstract: Interferon-gamma (IFN-gamma) exerts potent antiviral activity in the hepatitis C virus (HCV) replicon systems. However, the mechanisms underlying the direct antiviral effect have not been determined. We found that the type II transcriptional response to IFN-gamma could be suppressed by inhibition of MEK1/2 kinase activity by MEK1/2 inhibitor U0126 in the hepatoma cell line Huh-7. Using a bicistronic HCV replicon system expressing a luciferase reporter gene in Huh-7 cells (RLuc-replicon), we showed that inhibition of MEK1/2 kinase activity is sufficient to counteract the antiviral activity of IFN-gamma. Expression of a constitutive active form of Ras inhibited the luciferase activity of RLuc-replicon, whereas a dominant-negative mutant of Ras enhanced the reporter activity, indicating that the Ras-MAPK pathway has a role in limiting replication of the viral RNA. Consistent with the involvement of the Ras-MAPK pathway, treatment with epidermal growth factor suppressed HCV protein expression in the RLuc-replicon cells, an effect that could be abolished by U0126. Inhibition of MEK1/2 kinase activity correlated with reduced phosphorylation of the HCV NS5A protein and enhanced RLuc-replicon luciferase reporter activity, in line with recent reports that phosphorylation of NS5A negatively modulates HCV RNA replication. Finally, genetic deletion analysis in yeast supported the role of a MEK-like kinase(s) in the regulation of NS5A phosphorylation. In conclusion, the direct anti-HCV effect of IFN-gamma in cell culture is, at least in part, mediated through the Ras-MAPK signaling pathway, which possibly involves a direct or indirect modulation of NS5A protein phosphorylation.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.forename: Xinyi Cynthia
authors.initials: XC
authors.lastname: Chen
authors.forename: Madhavi
authors.initials: M
authors.lastname: Konduri
authors.forename: Nadejda
authors.initials: N
authors.lastname: Fomina
authors.forename: Jin
authors.initials: J
authors.lastname: Lu
authors.forename: Ling
authors.initials: L
authors.lastname: Jin
authors.forename: Alexander
authors.initials: A
authors.lastname: Kolykhalov
authors.forename: Seng-Lai
authors.initials: SL
authors.lastname: Tan
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1 / Q000502:physiology; D048370:MAP Kinase Kinase 2 / Q000502:physiology; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D018631:ras Proteins / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D007371:Interferon-gamma; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D017361:Viral Nonstructural Proteins; D018631:ras Proteins; C482128:MAP2K2 protein, human; C482125:MAP2K1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase