Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)
Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)
复制标题
丙型肝炎病毒 NS5B 蛋白通过重新定位细胞周期蛋白依赖性激酶 2 相互作用蛋白 (CINP) 延迟人肝细胞来源细胞的 S 期进展
DOI：
10.1074/jbc.m111.225672
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发表时间：
2011-07-29
影响因子：
4.8
通讯作者：
Yuan, Zhenghong
中科院分区：
文献类型：
--
作者：
Wang, Yaohui;Wang, Yuchan;Yuan, Zhenghong
细胞周期失调是病毒感染相关肿瘤发生的关键事件。先前的研究表明，丙型肝炎病毒NS5B除了作为RNA依赖的RNA聚合酶参与RNA合成外，还可以调节细胞周期进程。然而，分子机制至今仍不清楚。本研究通过建立HepG2 Tet-On NS5B稳定细胞系来证实NS5B对细胞周期的影响。为了更好地了解NS5B在细胞周期调控中的作用，利用人肝脏cDNA文库进行了酵母双杂交实验。鉴定了周期蛋白依赖性激酶2相互作用蛋白（CINP）。体内和体外实验进一步证实了NS5B和CINP之间的相互作用，并且发现它们的关联对于S期延迟和细胞增殖抑制是必不可少的。进一步的实验表明，NS5B将CINP从细胞核重新定位到细胞质。通过特异性siRNA直接敲除CINP导致DNA损伤反应和细胞周期检查点蛋白表达的显著改变，包括p21的增加和磷酸化的视网膜母细胞瘤和Chk1的减少。在表达NS5B的细胞中观察到类似的结果，并且对异位过表达CINP的影响部分逆转。这些研究表明，DNA损伤反应可能被NS5B利用来阻碍细胞周期的进展。综上所述，我们的数据表明NS5B通过与CINP的相互作用以及蛋白质从细胞核到细胞质的重新定位而延迟细胞进入S期。这些作用可能有助于丙型肝炎病毒的持久性和发病机制。
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.