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载体的血液样本中构建了乙型肝炎病毒（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。细胞系对抗FAS单克隆抗体的刺激显示凋亡变化。用抗体处理的细胞显示了广泛的细胞圆形，收缩和细胞质吹动，并最终从板中脱离。在细胞核中观察到染色质的碎裂，并检测到DNA梯子。通过用特异性抑制剂的半胱氨酸蛋白CPP32预处理来预防凋亡细胞死亡。白介素1b转换酶的特定抑制剂并未显示预防效应。结果表明（i）HCV核心蛋白的细胞内表达使细胞容易出现凋亡死亡，而不会上调表面FAS表达，并且（ii）CPP32蛋白在HCV核心表达细胞的凋亡效应途径中起着作用。 HCV Core Prot…更多的EIN可能在免疫介导的肝细胞损伤中起作用。为了建立一种在哺乳动物细胞中产生小型HCV的系统，我们在CAG启动器的控制下（AdexCatCatCatcat7）构建了表达细菌T7 RNA RNA聚合酶的复制性重组腺病毒T7 RNA聚合酶。感染ADEXCAT7后，在T7启动子的控制下用携带报告基因荧光素酶基因的质粒翻译了各种细胞系。荧光素酶的表达高度在人类肝细胞癌的细胞系中。在该细胞系中，还通过在体外合成的RNA转染来观察到较高水平的报告基因表达。该混合T7腺病毒系统可以在体内合成HCV微型元素，可能有助于阐明人肝细胞中HCV复制的分子机制。HCV的核心蛋白预计与病毒sense RNA结合而成形成核OCAPSID，因为其碱性氨基含量的高含量是碱性氨基含量的。阐明结合特性以了解HCV的复制机制非常重要。我们在体外合成了HCV病毒和抗病毒RNA的各个区域，并转化为表达HCV核心蛋白的HEPG2细胞。用抗核抗体免疫沉淀细胞裂解物。从核心蛋白和RNA的免疫沉淀物中提取RNA，并通过DOT印迹或North blot分析确定RNA的极性。核心蛋白的特异性结合被观察到一定长度的阳性HCV RNA，但没有负面的含义。病毒感官RNA的缺失分析表明，基因组RNA的381NT（329-710NT）（329-710nt）负责与核心蛋白的特异性结合。此外，我们准备了缺乏核心蛋白中每个碱性氨基酸簇的核心蛋白的缺失突变体。核心蛋白中的14AA的碱性氨基酸簇在与病毒RNA结合中起重要作用。较少的

项目成果

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。”

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）。

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）。

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）。