Elucidation of the molecular mechanism of chronic hepatitis through the analysis of intracellular function of NS5A protein of hepatitis C virus

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Suppression of intracellular interferon signaling pathway by NS5A protein: The aim of this study was to examine the effect of the NS5A protein on cellular responses to IFN. For the expression of the NS5A protein, the entire NS5A region of HCV, that have either the mutant or the wild type ISDR sequences, were cloned into the pCI-neo mammalian expression vector or into an adenovirus vector. For the analysis of IFN signal transduction, IFN inducible reporter plasmids were constructed in which luciferase reporter were driven by the promoter region of IFN-responsive gene 6-16 or by the consensus motif of the IFN stimulatoryresponse element (ISRE). These reporter plasmids were transfected to HepG2 or Huh-7 cells expressing the NS5A protein. The induction of luciferase activity by IFN stimulation was 10 to 45% repressed in cells expressing the NS5A protein compared to those transfected with the mock plasmid. This inhibitory effect was more prominent with the NS5A protein carring the wild type ISDR sequence than those having one to seven amino acid mutations. These results provide a new mechanism for inhibition of the IFN-alpha-activated antiviral response by the NS5A protein and suggests that NS5A has functional significance for IFN resistance.Suppression of TNF mediated apoptosis by NS5A protein: It is known that serum level of HCVRNA declines in a biphasic way, and the second phase decline is thought to be mediated by apoptotic elimination of HCV infected cells. Thus, we evaluated the effect of NS5A protein on the TNF alphamediated apoptosis. As a result, apoptosis was repressed in a huh7 cell expressing Huh7 cell. The activity of caspase 3, 9, 8 was all decreased. Apoptosis induced by forced expression of caspase 8 was not blocked by NS5A suggesting that inhibitory action of NS5A is targetted upstream of caspase 8.