Enzymatic Differences Among Hepatitis C Virus Genotypes

描述（由申请人提供）：几乎每五十五个美国人中就有一个人接触过丙型肝炎病毒（HCV），但大多数人都不知道自己感染了，因为这种病毒很少引起急性症状。如果不治疗，大多数HCV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。目前使用干扰素和利巴韦林等药物的治疗费用昂贵，而且会产生使人衰弱的副作用，通常比HCV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非常有效。该提案将检查最直接参与病毒复制的HCV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抗病毒剂的靶点。除了其作为免疫系统调节剂的既定作用外，利巴韦林已被提议作为诱变剂或通过对病毒复制蛋白的直接作用来消除病毒。一种流行的假设认为，利巴韦林对已经很高的HCV突变率的增强导致了错误的灾难和随后的病毒消除。在这里，利巴韦林的影响将在体外，在酶测定，并在体内，使用一种新的HCV复制子，应允许复制保真度的评估。为了尝试将利巴韦林的作用与基因型特异性药物反应联系起来，所有实验将用三种最常见的美国HCV基因型重复，两种通常对治疗无反应（Ia和Ib），一种经常对治疗有反应（2a）。还将表征来自每个基因型的聚合酶和解旋酶蛋白以定义保守和不同的性质。严格的生物化学方法将用于定义酶的差异，因为单独的序列数据不能准确预测蛋白质的结构或功能。初步数据显示，不同基因型编码的酶具有明显不同的特性，阻碍了目前合理的药物设计工作。基于结构的定点突变将用于确定HCV酶变异性的遗传基础。然后将在复制子系统中分析HCV遗传变异的生物学后果（即复制率、保真度、蛋白质表达）。对某些表型的遗传变异的描述可能有助于预测患者对当前或未来HCV治疗的反应，而对保守HCV酶特性的明确鉴定将有助于未来HCV药物的开发。

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.