Study on maturation and replication mechanisms of Pestivirus and hepatitis C virus

瘟病毒和丙型肝炎病毒的成熟和复制机制研究

• 批准号: 07456137
• 负责人: MATSUURA Yoshiharu
• 金额: $ 4.1万
• 依托单位: National Institute of Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456137/
• 关键词: HCV,; Gene Expression,; Apoptosis; ペスティウイルス; ウイルスの成熟機構

We constructed a full length cDNA clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier and established a HepG2 cell line constitutively expressing HCV core protein. The cell line showed apoptotic changes in response to stimulation with anti-Fas monoclonal antibody. Cells treated with the antibody showed extensive cell rounding, shrinkage and cytoplasmic blebbing, and finally detached from plates. Fragmentation of the chromatin was observed in the nucleus and DNA ladders were detected. Apoptotic cell death was prevented by pretreatment with a specific inhibitor of the cysteine protease CPP32. While the specific inhibitor of interleukin-1b-converting enzyme did not show the preventive effect. The results suggest (i) that intracellular expression of HCV core protein makes cells prone to apoptotic death without upregulation of surface Fas expression, and (ii) that the CPP32 protease plays a part in the apoptosis effector pathway of HCV core expressing cells. HCV core prot … More ein may have a role in immune mediated liver cell injury.To establish a system to produce minigene RNA of HCV in mammalian cells, we constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). After infection with AdexCAT7, various cell lines were transfected with plasmids carrying reporter luciferase gene under the control of T7 promoter. Expression of luciferase was highest in a cell line derived from human hepatocellular carcinoma. In this cell line, higher level of reporter gene expression was also observed by transfection of RNA synthesized in vitro. This hybrid-T7 adenovirus system to synthesize HCV minigenes in vivo may be useful to elucidate the molecular mechanism of HCV replication in human liver cells.The core protein of HCV is expected to bind with viral sense RNA to form a nucleocapsid because of its high content of basic amino acid residues. It is very important to clarify the binding property to understand the replication mechanism of HCV.We synthesized various regions of viral and anti-viral RNA of HCV in vitro and transfected into HepG2 cells expressing HCV core protein. Cell lysates were immunoprecipitated with anti-core antibody. RNA was extracted from the immunoprecipitates of core protein and RNA,and polarity of the RNA was determined by dot blot or northern blot analysis. Specific binding of core protein was observed with a full length of positive sense HCV RNA,but not with negative sense. Deletion analysis of viral sense RNA revealed that 381nt (329-710nt from the 5'end) of the genomic RNA is responsible for specific binding with core protein. Furthermore, we prepared deletion mutants of core protein lacking each of basic amino acid clusters in core protein. A basic amino acid cluster of 14aa in core protein was shown to play an important role in binding with the viral RNA. Less

我们从一名丙型肝炎病毒携带者的血液样本中构建了丙型肝炎病毒的全长cDNA克隆，并建立了组成性表达丙型肝炎病毒核心蛋白的HepG2细胞系。细胞株在抗Fas单抗刺激下出现明显的细胞凋亡改变。经该抗体处理的细胞出现广泛的细胞圆形、皱缩和胞浆气泡，最后从平板上脱落。核染色质断裂，DNA梯状条带出现。用半胱氨酸蛋白酶CPP32的特定抑制剂进行预处理，可防止细胞凋亡。而白介素1b转换酶的特异性抑制剂并未显示出预防作用。结果提示：(1)细胞内表达的丙型肝炎病毒核心蛋白可使细胞发生凋亡，而表面Fas表达不上调；(2)CPP32蛋白参与了丙型肝炎病毒核心蛋白表达细胞的凋亡效应途径。丙型肝炎病毒核心蛋白…为了建立在哺乳动物细胞中产生丙型肝炎病毒微小基因RNA的系统，我们构建了在CAG启动子控制下表达噬菌体T7 RNA聚合酶的复制缺陷重组腺病毒(AdexCAT7)。感染AdexCAT7后，在T7启动子的控制下，将报告基因荧光素酶基因导入不同的细胞系。荧光素酶在人肝细胞癌细胞系中的表达最高。在该细胞系中，体外合成的RNA也能获得较高水平的报告基因表达。该混合T7腺病毒系统可在体内合成丙型肝炎病毒的微小基因，可能有助于阐明丙型肝炎病毒在人肝细胞内复制的分子机制。丙型肝炎病毒的核心蛋白由于含有较高的碱性氨基酸残基，有望与病毒正义RNA结合形成核衣壳。我们在体外合成了不同区域的丙型肝炎病毒核糖核酸，并将其导入表达丙型肝炎病毒核心蛋白的HepG2细胞中。细胞裂解物与抗核抗体免疫共沉淀。从核心蛋白和RNA的免疫沉淀物中提取RNA，通过斑点印迹或Northern印迹分析确定RNA的极性。发现核心蛋白与全长的正义型丙型肝炎病毒RNA特异性结合，而不与负义型丙型肝炎病毒RNA结合。病毒正义RNA的缺失分析表明，基因组RNA的381nt(从5‘端起329-710nt)负责与核心蛋白的特异性结合。此外，我们还制备了缺失每个核心蛋白碱性氨基酸簇的核心蛋白缺失突变体。核心蛋白中一个14aa的碱性氨基酸簇在与病毒RNA的结合中起着重要作用。较少

项目成果

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