Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner

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Abstract
We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (−/−) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.