Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis worldwide and may lead to the development of hepatocellular carcinoma. However, the mechanism of development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. In the present study, we have investigated the effect of nonstructural protein 5A (NS5A) on TNF- and Fas-mediated apoptosis in the liver of transgenic mice. For this purpose, transgenic mice were generated by targeting the HCV NS5A genomic region cloned under the control of a liver-specific apoE promoter. The transgenic animals were phenotypically similar to their normal littermates and did not exhibit a detectable histological change in the liver at 8-12 weeks of age. Intraperitoneal injection of recombinant TNF induced hepatic injury and apoptosis in normal mice. In contrast, transgenic mice expressing NS5A protein were protected against hepatic apoptosis after injection of TNF. However, injection of anti-Fas antibody into transgenic mice did not significantly influence hepatic apoptosis compared to the normal littermates. These results suggested distinct effects of TNF and anti-Fas antibody in transgenic mice expressing NS5A. We subsequently investigated the effect of NS5A in signaling pathways involved in these two cytokine-mediated apoptosis. A physical association between NS5A and TRADD was observed by pull-down assay, coimmunoprecipitation, and colocalization experiments. Furthermore, NS5A prevented the association between TRADD and FADD and blocked TRADD-mediated NF-κB activation. Together, our results suggest that NS5A impairs TNF-mediated apoptosis by interfering upstream of the signal transduction pathway and may play a role in HCV-mediated pathogenesis.
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