THE MECHANISMS OF ANTI-APOPTOTIC EFFECTS BY HEPATITIS C VIRUS CORE PROTEIN

为阐明丙型肝炎病毒（HCV）致肝炎及肝细胞癌的分子机制，本课题分析了HCV核心蛋白抗凋亡作用的机制。所取得的成就如下：分析了HCV核心蛋白活化细胞转录因子NF-κB的分子机制，认为核心蛋白活化细胞转录因子NF-κB是HCV核心蛋白抗凋亡作用的重要机制之一。如下图所示；(1)通过缺失分析和亚细胞定位观察，揭示了HCV核心蛋白在细胞质，特别是内质网膜上的定位对NF-κB的激活具有重要意义。(2)我们发现，与真正的核心蛋白一样，NF-κB激活所需的最小区域位于HCV核心蛋白的氨基末端aa20 ~ aa80。(3)以酵母双杂交系统获得的几个cDNA克隆为诱饵，对其进行了鉴定。(4)我们发现，核心蛋白可以增强IKKβ （IKKβ是一种磷酸化i -κB的激酶）过表达诱导的NF-κB活性，而不增强IKKβ活性，这表明核心蛋白可以影响NF-κB激活途径中IKKβ的下游点，从而增强NF-κB活性。我们分析了核心蛋白激活MAP激酶途径的分子机制，得到的结果表明，核心蛋白通过未知的机制增强转录因子Elk1的活性，这与已知的激活反应不同。由于HCV核心蛋白激活转录因子似乎与抑制细胞凋亡、HCV持续感染和肝细胞癌变密切相关，目前正在进一步分析其分子机制。

To clarifiy the molecular mechanism of hepatitis and hepatocellular carcinoma caused by hepatitis C virus (HCV), we analysed the mechanism of anti-apoptotic effect by HCV core protein in this research project. The achievements are listed bellow.1. Results obtained from analysis of the molecular mechanism for activation of cellular transcription factor NF-κB by HCV core protein which is thought be one of important machinary for the anti-apoptotic effect of the core portein. are shown as follows ;(1) From th edeletion analysis and and the observation of subcellular localization, it was revealed that the localization of the HCV core protein in the cytoplasm, especially on the endoplasmic reticulum membranes, is important for the NF-κB activation. .(2) We found that the minimum region required for NF-κB activation just like as authentic core protein is located at the amino-terminal portion, from aa20 to aa80, of the HCV core protein.(3) We are trying to characterize several cDNA clones obtained by yeast two hybrid system using the region from aa 20 to aa 80 of the core as a bait.(4) We showed that the core protein enhanced the NF-κB activity induced by the overexpression of IKKβ, which is a kinase for the phosphorylation of IκB, without augmentation of IKKβ activity, suggesting that the core protein affects the point downstream of IKKβin thee NF-κB activation pathway to enhance the NF-κB activity.2. We analysed the molecular mechanism of activation of MAP kinase pathway by the core protein and obtained the results showing that the coree protein augments the activity of transcription factor Elk1 by the unknow mechaism which is different from the well-known kination reaction.Since the activation of the tramscription factors by the HCV core protein seems to be closely related with the suppression of apoptosis, persistent infection of HCV, and hepatocellular carcinogenesis, further analysis of molecular mechanisms is now underway.