Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)

Yaohui Wang,Yuchan Wang,Yan Xu,Wenyan Tong,Tingting Pan,Jianhua Li,Shuhui Sun,Junjie Shao,Huanping Ding,Tetsuya Toyoda,Zhenghong Yuan

doi:10.1074/jbc.m111.225672

Abstract

Cell cycle dysregulation is a critical event in virus infection-associated tumorigenesis. Previous studies have suggested that hepatitis C virus NS5B modulates cell cycle progression in addition to participating in RNA synthesis as an RNA-dependent RNA polymerase. However, the molecular mechanisms have thus far remained unclear. In this study, a HepG2 Tet-On NS5B stable cell line was generated to confirm the effect of NS5B on the cell cycle. To better understand the role of NS5B in cell cycle regulation, yeast two-hybrid assays were performed using a human liver cDNA library. The cyclin-dependent kinase 2-interacting protein (CINP) was identified. The interaction between NS5B and CINP was further demonstrated by in vivo and in vitro assays, and their association was found to be indispensable for S phase delay and cell proliferation suppression. Further experiments indicated that NS5B relocalized CINP from the nucleus to the cytoplasm. Directly knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Similar results were observed in cells expressing NS5B, and the effects were partially reversed upon ectopic overexpression of CINP. These studies suggest that the DNA damage response might be exploited by NS5B to hinder cell cycle progression. Taken together, our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm. Such effects might contribute to hepatitis C virus persistence and pathogenesis.

Highlights

At present, there are at least 170 million people infected with the hepatitis C virus (HCV)4 worldwide [1, 2], and the incidence increases yearly, especially in developing countries
We confirmed that NS5B induced cell cycle delay in S phase, and for the first time, we identified a novel human protein, cyclin-dependent kinase 2-interacting protein (CINP), that interacted with NS5B and was involved in the cell cycle dysfunction caused by NS5B
U-2 OS cells were transfected with NS5B-expressing plasmids or empty vectors for 48 h, and cell cycle distribution was analyzed by flow cytometry

Summary

Introduction

There are at least 170 million people infected with the hepatitis C virus (HCV)4 worldwide [1, 2], and the incidence increases yearly, especially in developing countries. Knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm.

Results

Conclusion