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（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。抗fas单克隆抗体刺激后，细胞出现凋亡变化。用抗体处理的细胞显示广泛的细胞圆缩、收缩和细胞质起泡，最后与板分离。在细胞核中观察到染色质的断裂，并检测到DNA阶梯。半胱氨酸蛋白酶CPP32特异性抑制剂预处理可防止凋亡细胞死亡。而白细胞介素-1b转换酶特异性抑制剂未显示出预防作用。结果表明：(1)细胞内表达HCV核心蛋白可使细胞发生凋亡死亡，而不上调表面Fas的表达；(2)CPP32蛋白酶参与HCV核心表达细胞的凋亡效应通路。丙型肝炎病毒核心蛋白在免疫介导的肝细胞损伤中可能起作用。为了建立在哺乳动物细胞中产生HCV小基因RNA的系统，我们构建了在CAG启动子（AdexCAT7）控制下表达噬菌体T7 RNA聚合酶的复制缺陷重组腺病毒。感染AdexCAT7后，在T7启动子的控制下，用携带报告荧光素酶基因的质粒转染不同细胞系。荧光素酶在人肝细胞癌细胞系中表达最高。在该细胞系中，通过转染体外合成的RNA也观察到更高水平的报告基因表达。这种在体内合成HCV微小基因的混合t7腺病毒系统可能有助于阐明HCV在人肝细胞内复制的分子机制。由于HCV核心蛋白的碱性氨基酸残基含量高，因此有望与病毒感RNA结合形成核衣壳。阐明其结合特性对了解丙型肝炎病毒的复制机制具有重要意义。我们在体外合成了HCV不同区域的病毒和抗病毒RNA，并转染到表达HCV核心蛋白的HepG2细胞中。细胞裂解液用抗核心抗体免疫沉淀。从核心蛋白和RNA的免疫沉淀物中提取RNA，用点印迹或northern印迹法测定RNA的极性。全长正义HCV RNA与核心蛋白特异性结合，而全长负义HCV RNA不与核心蛋白特异性结合。病毒感RNA的缺失分析显示，基因组RNA的381nt（5'端329-710nt）负责与核心蛋白的特异性结合。此外，我们制备了缺失核心蛋白中每个基本氨基酸簇的核心蛋白缺失突变体。核心蛋白中的一个碱性氨基酸簇14aa在与病毒RNA的结合中起重要作用。少

项目成果

Moriya K.: "Hepatitis C virus core protein induces hepatic steatosis in transgenic mice." J.Gen Virol.(印刷中).

Moriya K.：“丙型肝炎病毒核心蛋白诱导转基因小鼠肝脂肪变性。”（J.Gen Virol）。

Shoji I., Suzuki T., Chieda S., Sato M., Harada T., Chiba T., Matsuura Y., Miyamura T.: "Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli." Hepatology. 22. 1648-1655 (1995)

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。”

Yap C-C., Ishii K., Aoki Y., Aizaki H., Tani H., Shimizu H., Ueno Y., Miyamura T., Matsuura Y.: "A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA." Virology. (in press).

Yap C-C.、Ishii K.、Aoki Y.、Aizaki H.、Tani H.、Shimizu H.、Ueno Y.、Miyamura T.、Matsuura Y.：“用于恢复感染性病毒的混合杆状病毒-T7 RNA 聚合酶系统

Koike K.: "Sialadenitis resembling Sjogren's syndrome in hepatitis C virus transgenic mice." Proc.Natl.Acad.Sci.USA.(印刷中). (1997)

Koike K.：“丙型肝炎病毒转基因小鼠的唾液腺炎”，《美国国家科学院院刊》（1997 年）。

Ruggieri A.: "Sensitization to Fas-mediated Apoptosis by Hepatitis C Virus Core Protein." Virology. (印刷中). (1997)

Ruggieri A.：“丙型肝炎病毒核心蛋白对 Fas 介导的细胞凋亡的敏感性”（正在出版）。