Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein
Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein
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DOI：
10.1002/pmic.200401093
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发表时间：
2005-05-01
期刊：
影响因子：
3.4
通讯作者：
Oh, JW
中科院分区：
文献类型：
--
作者：
Kang, SM;Shin, MJ;Oh, JW
丙型肝炎病毒（HCV）是慢性肝炎和肝细胞癌的病原体。HCV的核心蛋白包装病毒RNA基因组以形成核衣壳。除了作为结构蛋白的功能外，核心蛋白还参与调节细胞转录、病毒诱导的转化和发病机制。为了通过鉴定与核心蛋白相互作用的细胞蛋白质来深入了解核心蛋白的细胞功能，我们采用了蛋白质组学方法。将肝细胞可溶性胞浆蛋白与Ni-次氮基三乙酸树脂固定的核心蛋白结合，通过双向电泳分离总结合细胞蛋白。基质辅助激光解吸/电离飞行时间质谱相互作用的蛋白质的分析允许识别14个细胞蛋白结合的核心蛋白。这些蛋白质包括DEAD盒多肽5，其功能与先前通过酵母双杂交筛选鉴定的已知蛋白质相似，以及13种新鉴定的细胞蛋白质。有趣的是，9个蛋白质点被鉴定为中间微丝蛋白，包括细胞角蛋白（5个点为细胞角蛋白8，2个为细胞角蛋白19，1个为细胞角蛋白18）和波形蛋白。细胞角蛋白8和波形蛋白，这是以前被证明是参与其他病毒的感染过程，进一步分析，以确认其在体内的相互作用与核心蛋白的免疫印迹和免疫荧光显微镜。我们讨论功能。核心蛋白与新鉴定的细胞蛋白在HCV感染和发病机制中的相互作用的意义。
Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional. implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.