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

从丙型肝炎病毒（HCV）携带者的血液样品中构建了HCV的全长cDNA克隆，并建立了组成型表达HCV核心蛋白的HepG 2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、收缩和细胞质起泡，并最终从板上脱离。在细胞核中观察到染色质片段化，并检测到DNA梯状条带。通过用半胱氨酸蛋白酶CPP 32的特异性抑制剂预处理来防止细胞凋亡。而白细胞介素-1b转换酶特异性抑制剂则无预防作用。结果表明（i）HCV核心蛋白的细胞内表达使细胞倾向于凋亡性死亡而不上调表面Fas表达，和（ii）CPP 32蛋白酶在HCV核心表达细胞的凋亡效应子途径中起作用。HCV核心蛋白 ...更多信息 为了建立在哺乳动物细胞中生产HCV小基因RNA的系统，我们构建了一个在CAG启动子控制下表达噬菌体T7 RNA聚合酶的复制缺陷型重组腺病毒（AdexCAT 7）。用AdexCAT 7感染后，用携带在T7启动子控制下的报告荧光素酶基因的质粒转染各种细胞系。荧光素酶的表达在来源于人肝细胞癌的细胞系中最高。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。HCV核心蛋白含有大量的碱性氨基酸残基，可与病毒正义RNA结合形成核衣壳，因此，利用T7腺病毒载体系统在体内合成HCV小基因，有助于阐明HCV在人肝细胞中复制的分子机制。阐明HCV的结合特性对理解HCV的复制机制具有重要意义。我们体外合成了HCV的病毒和抗病毒RNA的不同区域，并转染表达HCV核心蛋白的HepG 2细胞。用抗核心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，并通过斑点印迹或北方印迹分析确定RNA的极性。核心蛋白可与全长正义HCV RNA特异性结合，但与反义HCV RNA不结合。病毒正义RNA的缺失分析表明，病毒基因组RNA的381 nt（从5 '端开始的329- 710 nt）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碱性氨基酸簇的核心蛋白缺失突变体。核心蛋白的14个氨基酸组成的碱性氨基酸簇在与病毒RNA结合中起重要作用。少

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