Lan Keng-Hsin, Sheu Meei-Ling, Hwang Shinn-Jang, Yen Sang-Hue, Chen Shiow-Yi, Wu Jaw-Ching, Wang Yuan-Jan, Kato Naoya, Omata Masao, Chang Full-Young, Lee Shou-Dong
Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University School of Medicine, Taipei 11217, Taiwan, Republic of China.
Oncogene. 2002 Jul 18;21(31):4801-11. doi: 10.1038/sj.onc.1205589.
Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG- and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAF(II)32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAF(II)32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.
丙型肝炎病毒（HCV）可引起持续性感染、慢性肝炎和肝细胞癌。HCV NS5A是HCV的非结构蛋白之一，被认为在致癌转化中发挥作用。由于肿瘤抑制因子p53对预防肿瘤转化很重要，我们研究了HCV NS5A对p53的功能影响。采用体外和体内共免疫沉淀及共聚焦显微镜来确定NS5A与p53的相互作用。HCV NS5A直接与p53结合，并使p53在核周区域共定位。通过报告基因检测，NS5A以剂量依赖的方式抑制p53的转录反式激活。利用FLAG-和FLAG-NS5A Hep3B稳定细胞系证实，NS5A下调了Hep3B细胞中由p53激活的内源性p21/waf1表达。通过流式细胞术在永久和瞬时转染外源性p53的NS5A Hep3B细胞中测定NS5A对p53介导的凋亡的影响。NS5A消除了p53诱导的凋亡，且抑制作用与NS5A与p53的结合能力密切相关。此外，HCV NS5A蛋白在体内与TFIID的一个成分及p53的必需共激活因子hTAF(II)32相互作用并共定位。这些结果表明，HCV NS5A在细胞质中与p53和hTAF(II)32相互作用并部分隔离它们，在HCV感染期间抑制p53介导的转录反式激活和凋亡，这可能有助于HCV感染的肝癌发生。
