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HCV携带者的血液样本中构建了丙型肝炎病毒（HCV）的全长cDNA克隆，并建立了组成型表达HCV核心蛋白的HepG2细胞系，该细胞系在抗Fas单克隆抗体的刺激下表现出凋亡变化。用抗体处理的细胞显示出广泛的细胞变圆、皱缩和细胞质起泡，最后在细胞核和 DNA 中观察到染色质断裂。检测到细胞凋亡通过半胱氨酸蛋白酶 CPP32 的特异性抑制剂进行预处理，而白细胞介素 1b 转换酶的特异性抑制剂没有显示出预防作用。 HCV 核心蛋白的表达使细胞在不上调表面 Fas 表达的情况下容易发生凋亡性死亡，并且 (ii) CPP32 蛋白酶在 HCV 核心表达的凋亡效应途径中发挥作用HCV 核心蛋白可能在免疫介导的肝细胞损伤中发挥作用。为了建立在哺乳动物细胞中产生 HCV 小基因 RNA 的系统，我们构建了一种在 HCV 控制下表达噬菌体 T7 RNA 聚合酶的复制缺陷型重组腺病毒。 CAG启动子(AdexCAT7)感染AdexCAT7后，用携带报告荧光素酶基因的质粒转染，在T7启动子的控制下表达。荧光素酶在源自人肝细胞癌的细胞系中最高，通过转染体外合成的RNA也观察到较高水平的报告基因表达，这种杂交T7腺病毒系统在体内合成HCV小基因可能是有用的。阐明HCV在人肝细胞中复制的分子机制。HCV的核心蛋白由于其碱性氨基酸残基含量高，有望与病毒正义RNA结合形成核衣壳。阐明结合特性对于理解HCV的复制机制非常重要。我们在体外合成了HCV的病毒和抗病毒RNA的各个区域，并转染到表达HCV核心蛋白的HepG2细胞中，用抗核心蛋白对细胞裂解物进行免疫沉淀。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 blot分析观察核心蛋白的特异性结合，全长呈阳性。 HCV RNA，但不是病毒正义RNA的删除分析表明，基因组RNA的381nt（距5'端329-710nt）负责与核心蛋白的特异性结合。此外，我们制备了核心蛋白的删除突变体。核心蛋白中缺乏每个碱性氨基酸簇，表明核心蛋白中的14个氨基酸簇在与病毒RNA的结合中发挥重要作用。

项目成果

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Shoji I.、Suzuki T.、Chieda S.、Sato M.、Harada T.、Chiba T.、Matsuura Y.、Miyamura T.：“丙型肝炎病毒 NS3 丝氨酸蛋白酶在大肠杆菌中有效表达的蛋白水解活性。”