Induction of ERK1/2 phosphorylation by ribavirin correlated with p53 phosphorylation and suppression of HCV replication.

Abstract

<p>(<b>A</b>) Dose-dependent phosphorylation of ERK1/2 by ribavirin. HepG2 cells were treated for 24 h with the indicated doses of ribavirin. (<b>B</b>) The association of ribavirin-induced ERK1/2 phosphorylation with p53 phosphorylation in a time-course analysis. HepG2 cells were untreated or treated with 30 µg/ml ribavirin and then harvested at the indicated time points. In addition, we used small-interference RNA (siRNA) to knockdown ERK1/ERK2 to assess the role of ERK1/2 and p53 in the suppression of HCV replication. (<b>C</b>) Cells were transduced with ERK1/2-siRNA (100 nM) and scrambled-siRNA for 48 h and treated with ribavirin (100 µg/ml) for 24 h in HCV replicon cells (JFH1/HepG2) and HepG2 cells. Cells were lysed, and analyzed for the expression of phosphorylated ERK1/2 (ERK1/2∼<i>pi</i>), phosphorylated p53 at Ser15 (p53ser15∼<i>pi</i>), p53, ERK1/2, Mdm2 and HCV NS3 proteins by immunoblotting. (<b>D</b>) HCV RNA expression levels of scramble-siRNA- or si-ERK1/2-transduced cells with/without ribavirin treatment were determined using quantitative RT-PCR. Each result represents the mean ± s.e.m of 5 independent measurements and is considered statistically significant at <i>P</i><0.05. Control: no ribavirin treatment, RBV: ribavirin.</p