Enzymatic Differences Among Hepatitis C Virus Genotypes

丙型肝炎病毒基因型之间的酶学差异

• 批准号: 7012264
• 负责人: David N Frick
• 金额: $ 30.56万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012264
• 关键词: Enzymatic; Differences; Among; Hepatitis; Virus

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.

描述（由申请人提供）：几乎每五十五名美国人中就有一人接触过丙型肝炎病毒 (HCV)，但大多数人并不知道自己受到感染，因为该病毒很少引起急性症状。如果不及时治疗，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。目前涉及干扰素和利巴韦林药物的疗法价格昂贵，并且会产生使人衰弱的副作用，通常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。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型抗病毒药物的靶点。除了作为免疫系统调节剂的既定作用外，利巴韦林还被提议作为诱变剂或通过直接作用于病毒复制蛋白来消除病毒。一种流行的假设指出，利巴韦林会增强已经很高的 HCV 突变率，从而导致灾难性的错误和随后的病毒消除。在这里，将使用一种新型 HCV 复制子在体外、酶测定和体内检查利巴韦林的作用，该复制子应该可以评估复制保真度。为了尝试将利巴韦林效应与基因型特异性药物反应联系起来，所有实验都将用三种最常见的美国 HCV 基因型重复，其中两种通常对治疗没有反应（la 和 lb），另一种经常对治疗有反应（2a）。来自每个基因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差异，因为仅序列数据并不能准确预测蛋白质结构或功能。初步数据表明，不同的基因型编码的酶具有明显不同的特性，阻碍了当前合理的药物设计工作。基于结构的定点诱变将用于确定 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）。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 HCV 疗法的反应，并且清楚地识别保守的 HCV 酶特性将有助于未来的 HCV 药物开发。