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

在美国，丙型肝炎病毒（HCV）是主要的病原体 慢性病毒性肝炎的病原体。 大多数感染者 将发展为进行性肝病，可能包括肝硬化， 肝细胞癌对HCV的研究一直受到严重关注。 由于缺乏体外细胞培养系统， 适当的动物模型。 因此，针对HCV的有效疗法 还有待开发。 干扰素治疗目前是 唯一可用的治疗HCV感染的方法。 不幸的是，不到50 接受高剂量干扰素治疗的患者中， 根除HCV的持续应答。 可能的遗传基础 干扰素耐药性的研究已经在一个小的，高度 HCV-1b的NS 5A基因产物的保守结构域，称为 干扰素敏感性决定区（ISDR）。 人们认为 NS 5A可能通过与干扰素相互作用介导HCV对干扰素的抗性。 一种或多种干扰素诱导的细胞蛋白，所述细胞蛋白与 干扰素抗病毒反应。 私家侦探实验室证明， NS 5A直接与干扰素诱导的蛋白质相互作用 激酶，PKR，干扰素的抗病毒作用的主要介质。 重要的是，这种互动似乎需要减灾战略。 它 他们假设NS 5A负责介导HCV 对干扰素治疗的抗性，至少部分地，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 NS 5A与PKR。该提案的具体目标包括：（1）在- NS 5A-PKR相互作用的深度结构-功能分析。 NS5A 将构建缺失突变体， 使用mutD 5诱变引入NS 5A。 突变型NS 5A 将使用PKR的体外和体内测定来检查构建体 活性以及逆转PKR诱导的缓慢生长的能力 酵母中的表型。 (2)稳定表达NS 5A的Huh-1的建立 7个细胞系以检查NS 5A对PKR功能的影响， 活动，并获得有关的信息，生物作用， HCV感染细胞中的NS 5A。 稳定表达NS 5A的NIH 3 T3细胞 此外，还将建造一些线路，以研究NS 5A在以下方面的潜在作用： 转化和肿瘤发生。 (3)NS 5A的鉴定 磷酸化位点。 这些网站对NS 5A的重要性 将通过定点诱变检查功能。

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.