HEPATITIS C VIRUS AND INTERFERON--CONTROL OF PKR BY NS5A

在美国，丙型肝炎病毒(HCV)是主要的病因 慢性病毒性肝炎的制剂。大多数感染者 会发展成进行性肝病，可能包括肝硬变和 肝细胞癌。对丙型肝炎病毒的研究一直很严重 由于缺乏体外细胞培养系统和 合适的动物模型。因此，有效的抗丙型肝炎病毒疗法 都还有待开发。目前干扰素的治疗是 丙型肝炎病毒感染的唯一可用治疗方法。不幸的是，不到50人 在接受大剂量干扰素治疗的患者中，有百分比的患者显示出 根除丙型肝炎持续有效。一种可能的遗传基础 对于干扰素的抗药性已经被发现在一种小的、高度的 丙型肝炎病毒1b的NS5A基因产物的保守结构域，称为 干扰素敏感性决定区(ISDR)。据认为， NS5A可能通过相互作用介导丙型肝炎病毒对干扰素的耐药性 一种或多种干扰素诱导的细胞蛋白与 干扰素抗病毒反应。私家侦探实验室已经证明 NS5A直接与干扰素诱导的蛋白质相互作用 PKR，干扰素抗病毒作用的主要介体。 值得注意的是，这种互动似乎需要ISDR。它 他们的假设是NS5A负责调节丙型肝炎病毒 对干扰素治疗的抵抗，至少部分是通过直接的 与PKR的互动。为了验证这一假设，他们将使用 多方面的方法来描述和定义 NS5A，带PKR。这项建议的具体目标包括：(1)在- NS5A-PKR相互作用的深度结构-功能分析。NS5A 将构建缺失突变体和定点突变 通过突变D5诱变导入NS5A。突变型NS5A 将使用体外和体内检测PKR的方法来检测构建物 活性以及逆转PKR诱导的缓慢生长的能力 酵母菌的表型。(2)稳定表达NS5A的HuH- 7细胞株检测NS5A对PKR功能和细胞周期的影响 活动，并获得有关的信息，生物学作用 丙型肝炎病毒感染细胞中的NS5A。稳定表达NS5A的NIH 3T3细胞 还将建造线路，以检查NS5A在 转化和肿瘤发生。(3)NS5A的鉴定 体内的磷酸化位点。这些地点对NS5A的重要性 功能将通过定点突变进行检测。

In the United States, hepatitis C virus (HCV) is the major etiologic agent of chronic viral hepatitis. The majority of infected individuals will develop progressive liver disease, which may include cirrhosis and hepatocellular carcinoma. Research on HCV has been severely hampered by the lack of an in vitro cell culture system and an adequate animal model. As a result, effective therapies against HCV have yet to be developed. Treatment with interferon is currently the only available therapy for HCV infection. Unfortunately, less than 50 percent of patients treated with high doses of interferon show a sustained response with eradication of HCV. A possible genetic basis for interferon resistance has been identified in a small, highly conserved domain of the NS5A gene product of HCV-1b, termed the interferon sensitivity determining region (ISDR). It is thought that NS5A may mediate HCV resistance to interferon by interacting with one or more interferon-induced cellular proteins associated with the interferon antiviral response. The P.I. laboratory has demonstrated that NS5A directly interacts with the interferon-induced proteins kinase, PKR, a primary mediator of the antiviral effects of interferon. Significantly, the ISDR appears to be required for this interaction. It is their hypothesis that NS5A is responsible for mediating HCV resistance to interferon therapy, at least in part, through a direct interaction with PKR. To test this hypothesis, they will use a multifaceted approach to characterize and define the interaction of NS5A with PKR. Specific goals of this proposal include: (1) an in- depth structure-function analysis of the NS5A-PKR interaction. NS5A deletion mutants will be constructed and site-specific mutations introduced into NS5A using mutD5 mutagenesis. Mutant NS5A constructs will be examined using in vitro and in vivo assays for PKR activity as well as for ability to reverse the PKR-induced slow-growth phenotype in yeast. (2) Establishment of stable NS5A-expressing Huh- 7 cell lines to examine the effects of NS5A on PKR function and activity and to obtain information regarding, the biological role of NS5A in HCV-infected cells. Stable NS5A-expressing NIH 3T3 cell lines will also be constructed to examine the potential role of NS5A in transformation and tumorigenesis. (3) Identification of NS5A phosphorylation sites in vivo. The importance of such sites for NS5A function will be examined by site-directed mutagenesis.