Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk
Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk
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DOI：
10.1099/vir.0.83510-0
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发表时间：
2008-05-01
影响因子：
3.8
通讯作者：
Hotta, Hak
中科院分区：
文献类型：
--
作者：
Inubushi, Sachiko;Nagano-Fujii, Motoko;Hotta, Hak
丙型肝炎病毒（Hepatitis C virus，HCV）是肝细胞癌的主要致病因子。然而，致癌的确切机制尚未阐明。最近有报道，Syk，一种非受体蛋白酪氨酸激酶，在人乳腺癌中作为一种有效的肿瘤抑制剂发挥作用。本研究首先检查了HCV感染对体内Syk表达的可能影响。免疫组化分析显示，内源性Syk，否则是弥漫性表达在正常肝细胞的细胞质中，被定位在细胞膜附近的斑片状图案在HCV感染的肝细胞。然后检查HCV蛋白和Syk在人肝癌衍生的Huh-7细胞中的可能相互作用。免疫沉淀分析表明，NS 5A与Syk有很强的相互作用。缺失-突变分析显示NS 5A的N-末端部分（aa 1-175）参与与Syk的物理相互作用。体外激酶测定表明，NS 5A抑制Syk的酶活性，并且除了N-末端175个残基之外，抑制Syk还需要NS 5A的中心部分（aa 237-302）。此外，Syk介导的磷脂酶C-γ 1的磷酸化被NS 5A下调。NS 5A与Syk的相互作用也在携带HCV RNA复制子或感染HCV的Huh-7.5细胞中检测到。总之，这些结果表明，NS 5A与Syk相互作用，导致其激酶活性的负调节。结果表明，NS 5A可能通过抑制Syk激酶活性参与肝细胞癌变。
Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma 1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.