Enzymatic Differences Among Hepatitis C Virus Genotypes

描述(申请人提供)：几乎每55个美国人中就有一个接触过丙型肝炎病毒(丙型肝炎病毒)，但大多数人不知道自己感染了丙型肝炎病毒，因为病毒几乎不会引起急性症状。如果不治疗，大多数丙型肝炎病毒感染会导致慢性活动性肝炎，最终发展为肝硬化、癌症或肝功能衰竭。目前涉及干扰素和利巴韦林的药物治疗费用高昂，而且会产生令人衰弱的副作用，往往比丙型肝炎病毒本身产生的症状更严重。然而，较新的治疗方法对某些病毒基因型相当有效。这项提案将研究与病毒复制最直接相关的丙型肝炎病毒蛋白，NS3解旋酶和NS5B聚合酶，作为药物利巴韦林的假定靶点和新的抗病毒药物的靶标。除了作为免疫系统调节器的既定角色外，利巴韦林还被提出作为诱变剂或通过直接作用于病毒复制蛋白来消除病毒。一种流行的假设说，利巴韦林提高了已经很高的丙型肝炎病毒变异率，导致了错误的灾难和随后的病毒消除。在这里，利巴韦林效应将在体外，酶分析和体内，使用一种新的丙型肝炎病毒复制子，应该允许评估复制保真度。为了试图将利巴韦林效应与特定的基因型别药物反应联系起来，所有实验都将以美国最常见的三种丙型肝炎病毒重复进行，其中两种通常对治疗无效(1a和1b)，另一种经常对治疗有反应(2a)。每种类型的聚合酶和解旋酶蛋白也将被表征，以定义保守和不同的特性。将使用严格的生化方法来定义酶的差异，因为单靠序列数据并不能准确预测蛋白质的结构或功能。初步数据显示，不同的基因类型编码的酶具有明显不同的性质，阻碍了当前合理的药物设计努力。基于结构的定点突变将被用来确定丙型肝炎病毒酶变异的遗传基础。然后将在复制子系统中分析丙型肝炎病毒基因变异的生物学后果(即复制率、保真度、蛋白表达)。描述导致某些表型的遗传变异可能有助于预测患者对当前或未来的丙型肝炎治疗的反应，而对保守的丙型肝炎病毒酶特性的明确识别将有助于未来的丙型肝炎药物开发。

DESCRIPTION (provided by applicant): Almost one in every fifty-five Americans have been exposed to the Hepatitis C Virus (HCV), but most are unaware of their infection because the virus causes few acute symptoms. If left untreated, the majority of HCV infections lead to chronic active hepatitis that eventually progresses to cirrhosis, cancer, or liver failure. Current therapies involving the drugs interferon and ribavirin are costly and produce debilitating side effects, frequently worse than the symptoms produced by HCV itself. Newer treatments are, however, quite effective against certain viral genotypes. This proposal will examine the HCV proteins most directly involved in viral replication, the NS3 Helicase and NS5B polymerase, as putative targets for the drug ribavirin and as targets for new antiviral agents. In addition to its established role as a modulator of the immune system, ribavirin has been proposed to eliminate viruses as a mutagen or through direct effects on viral replicative proteins. One popular hypothesis states that ribavirin's enhancement of the already high HCV mutation rate leads to a catastrophe of errors and subsequent virus elimination. Here, ribavirin effects will be examined in vitro, in enzyme assays, and in vivo, using a novel HCV replicon that should allow the assessment of replication fidelity. To attempt to relate ribavirin effects to genotype-specific drug response, all experiments will be repeated with the three most common American HCV genotypes, two that normally do not respond to therapy (la and lb) and one that frequently responds to therapy (2a). The polymerase and helicase proteins from each genotype will also be characterized to define conserved and divergent properties. A rigorous biochemical approach will be used to define enzyme differences because sequence data alone does not accurately predict protein structure or function. Preliminary data show that different genotypes encode enzymes with markedly different properties, hampering current rational drug design efforts. Structure-based site-directed mutagenesis will be used to determine the genetic basis for HCV enzyme variability. The biological consequences (i.e. replication rate, fidelity, protein expression) of HCV genetic variation will then be analyzed in a replicon system. The delineation of genetic variations responsible for certain phenotypes might allow the prediction of patient response to current or future HCV therapies, and the clear identification of conserved HCV enzyme properties will aid future HCV drug development.