title: Hepatitis C virus NS5B protein delays s phase progression in human hepatocyte-derived cells by relocalizing cyclin-dependent kinase 2-interacting protein (CINP).
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.
authors.affiliation: Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Shanghai 200032, China.
authors.forename: Yaohui
authors.initials: Y
authors.lastname: Wang
authors.forename: Yuchan
authors.initials: Y
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Wenyan
authors.initials: W
authors.lastname: Tong
authors.forename: Tingting
authors.initials: T
authors.lastname: Pan
authors.forename: Jianhua
authors.initials: J
authors.lastname: Li
authors.forename: Shuhui
authors.initials: S
authors.lastname: Sun
authors.forename: Junjie
authors.initials: J
authors.lastname: Shao
authors.forename: Huanping
authors.initials: H
authors.lastname: Ding
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D006367:HeLa Cells; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D002352:Carrier Proteins; D000071877:Checkpoint Kinase 1; D011494:Protein Kinases; D016160:Retinoblastoma Protein; D017361:Viral Nonstructural Proteins; C510853:CINP protein, human; C000605867:CHEK1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase