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Elucidation of the molecular mechanism of chronic hepatitis through the analysis of intracellular function of NS5A protein of hepatitis C virus

丙型肝炎病毒NS5A蛋白胞内功能分析阐明慢性肝炎的分子机制

基本信息

批准号：
13670499
负责人：
KUROSAKI Masayuki
金额：
$ 2.3万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670499/
关键词：
NS5A HCV Interferon apoptosis TNF alpha signal transduction chronic hepatitis

项目摘要

Suppression of intracellular interferon signaling pathway by NS5A protein: The aim of this study was to examine the effect of the NS5A protein on cellular responses to IFN. For the expression of the NS5A protein, the entire NS5A region of HCV, that have either the mutant or the wild type ISDR sequences, were cloned into the pCI-neo mammalian expression vector or into an adenovirus vector. For the analysis of IFN signal transduction, IFN inducible reporter plasmids were constructed in which luciferase reporter were driven by the promoter region of IFN-responsive gene 6-16 or by the consensus motif of the IFN stimulatoryresponse element (ISRE). These reporter plasmids were transfected to HepG2 or Huh-7 cells expressing the NS5A protein. The induction of luciferase activity by IFN stimulation was 10 to 45% repressed in cells expressing the NS5A protein compared to those transfected with the mock plasmid. This inhibitory effect was more prominent with the NS5A protein carring the wild type ISDR sequence than those having one to seven amino acid mutations. These results provide a new mechanism for inhibition of the IFN-alpha-activated antiviral response by the NS5A protein and suggests that NS5A has functional significance for IFN resistance.Suppression of TNF mediated apoptosis by NS5A protein: It is known that serum level of HCVRNA declines in a biphasic way, and the second phase decline is thought to be mediated by apoptotic elimination of HCV infected cells. Thus, we evaluated the effect of NS5A protein on the TNF alphamediated apoptosis. As a result, apoptosis was repressed in a huh7 cell expressing Huh7 cell. The activity of caspase 3, 9, 8 was all decreased. Apoptosis induced by forced expression of caspase 8 was not blocked by NS5A suggesting that inhibitory action of NS5A is targetted upstream of caspase 8.

NS5A蛋白抑制细胞内干扰素信号通路：本研究的目的是研究NS5A蛋白对细胞对干扰素反应的影响。为了表达NS5A蛋白，将含有突变型或野生型ISDR序列的整个丙型肝炎病毒NS5A区克隆到pCI-neo哺乳动物表达载体或腺病毒载体中。为了分析干扰素信号转导，构建了由干扰素反应基因6-16启动子区域或干扰素刺激反应元件(ISRE)的共有基序驱动的荧光素酶报告基因的干扰素可诱导报告质粒。将这些报告载体分别导入表达NS5A蛋白的HepG2或Huh-7细胞。在表达NS5A蛋白的细胞中，干扰素刺激诱导的荧光素酶活性比转染模拟质粒的细胞抑制了10%~45%。当NS5A蛋白携带野生型ISDR序列时，这种抑制作用比具有1到7个氨基酸突变的NS5A蛋白更明显。这些结果为NS5A蛋白抑制干扰素α激活的抗病毒反应提供了一种新的机制，并提示NS5A蛋白在干扰素抵抗中具有功能意义。NS5A蛋白抑制肿瘤坏死因子介导的细胞凋亡：已知血清中HCVRNA水平呈双相下降，第二阶段下降被认为是通过消除感染丙型肝炎病毒的细胞的凋亡来实现的。因此，我们评价了NS5A蛋白在肿瘤坏死因子α介导的细胞凋亡中的作用。结果，在表达Huh7的Huh7细胞中，细胞凋亡受到抑制。Caspase3、9、8活性均降低。NS5A不能阻断强制表达caspase8诱导的细胞凋亡，提示NS5A的抑制作用主要针对caspase8上游。