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

NS5A蛋白对细胞内干扰素信号通路的抑制：本研究的目的是研究NS5A蛋白对细胞对IFN反应的影响。为了表达NS5A蛋白，将HCV中具有突变型或野生型ISDR序列的整个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IFN信号转导，构建了IFN诱导报告质粒，其中荧光素酶报告质粒由IFN反应基因6-16的启动子区域或IFN刺激反应元件（ISRE）的共识基序驱动。将这些报告质粒转染到表达NS5A蛋白的HepG2或Huh-7细胞中。与转染模拟质粒的细胞相比，在表达NS5A蛋白的细胞中，IFN刺激诱导的荧光素酶活性被抑制了10 - 45%。这种抑制作用在携带野生型ISDR序列的NS5A蛋白中比携带1 ~ 7个氨基酸突变的NS5A蛋白更为明显。这些结果为NS5A蛋白抑制IFN α激活的抗病毒应答提供了新的机制，提示NS5A在IFN耐药中具有功能意义。NS5A蛋白抑制TNF介导的细胞凋亡：已知血清HCVRNA水平呈双期下降，第二期下降被认为是通过消除HCV感染细胞的凋亡介导的。因此，我们评估了NS5A蛋白在TNF α介导的细胞凋亡中的作用。结果表明，表达huh7的huh7细胞的凋亡受到抑制。caspase 3、9、8活性均降低。强迫表达caspase 8诱导的细胞凋亡未被NS5A阻断，提示NS5A的抑制作用是针对caspase 8上游。

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.