Dynamics of Hepatis C viral RNA-dependent RNA replication

描述（由申请人提供）：丙型肝炎病毒感染约3%的世界人口，包括美国的4-5百万。慢性感染导致肝硬化和癌症，2007年，HCV的死亡率超过了HIV。预期HCV感染的成功治疗将需要联合治疗，类似于目前对HIV感染的治疗，并且HCV RNA依赖性RNA聚合酶（NS 5 B）的抑制剂将是该治疗的基石。FDA最近批准了一种基于第一种直接抗病毒核苷类似物的新治疗方法，目前正在开发新的潜在非核苷抑制剂（NNI）。这些药物是通过使用基于在人肝癌细胞系中自我复制的亚基因组复制子的筛选而开发的。然而，酶活性的生物化学筛选受到限制，因为体外RNA合成的低效从头起始和病毒聚合酶不能结合来自溶液的双链体RNA（引物/模板），并且通常认为NS 5 B的所有晶体结构都处于失活状态。目前的酶测定呈现出缓慢起始动力学和快速延伸的未解决的混合物，因此，不可能知道给定的药物是否抑制起始或延伸。缺乏关于每类药物的结合亲和力和作用机制的定量数据。有必要对酶功能进行定量测定，以建立核苷酸掺入，延伸和核苷酸依赖性切除的动力学参数，我们最近发现的反应可以有效地去除核苷类似物。已经发现非核苷抑制剂（NNI）结合到聚合酶上的至少四个不同位点。这些关于各种抑制剂的数据提高了 关于不同NNI与不同酶位点结合的作用机制的重要问题。我们已经建立了有效的形成和纯化的活性，高度进行性的延伸复合物的条件，克服了详细的生化分析NS 5 B催化复制的主要障碍。在本提案中，我们将使用最先进的单转换动力学方法：（1）建立控制同源和非同源碱基对掺入的保真度和基线动力学参数;（2）检查核苷酸类似物掺入、延伸和切除的动力学;（3）建立每类非核苷抑制剂的作用模式;（4）量化耐药突变的影响。此外，氢/氘交换的研究将揭示酶的灵活性的变化，在从非活性到活性酶的过渡，我们将试图确定的延伸复合物的晶体结构。这项工作奠定了基础，了解结构/功能关系的RNA依赖的RNA聚合，目前正在研究的各种药物的作用机制，以及耐药性的演变。

DESCRIPTION (provided by applicant): The hepatitis C virus infects approximately 3% of the world's population, including 4-5 million in the USA. Chronic infection leads to liver cirrhosis an cancer, and in 2007, HCV surpassed HIV in mortality rates. It is expected that successful treatment of HCV infections will require a combination therapy, analogous to current treatments for HIV infections, and that inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) will be a cornerstone of that treatment. The FDA has recently approved a new treatment based upon the first direct antiviral nucleoside analog and new potential nonnucleoside inhibitors (NNI's) are currently in the pipeline. These pharmaceuticals have been developed by using screens based on subgenomic replicons that self- replicates in human hepatoma cell lines. However, biochemical screens for enzyme activity have been limited because of the inefficient de novo initiation of RNA synthesis in vitro and the inability of the viral polymerase to bind duplex RNA (primer/template) from solution, and it is commonly accepted that all crystal structures of NS5B are of an inactive state. Current enzyme assays present an unresolved mixture of slow initiation kinetics and fast elongation and, therefore, it is not possible to know whether a given drug inhibits initiation or elongation. Quantitative data on binding affinity and mechanism of action of each class of drugs are lacking. There is a need for a quantitative assay for enzyme function to establish the kinetic parameters governing nucleotide incorporation, extension and nucleotide-dependent excision, a reaction that we recently showed can effectively remove nucleoside analogs. Non-nucleoside inhibitors (NNI's) have been discovered that bind to at least four distinct sites on the polymerase. These data on various inhibitors raise important questions regarding the mechanisms of action of the different NNI's binding to distinct enzyme sites. We have established conditions for efficient formation and purification of an active, highly processive elongation complex, overcoming the major obstacle to detailed biochemical analysis of NS5B-catalyzed replication. In this proposal, we will use state of the art single turnover kinetic methods to: (1) Establish the fidelity and baseline kinetic parameters governing cognate and noncognate base pair incorporation; (2) Examine the kinetics of incorporation, extension and excision of nucleotide analogs; (3) Establish modes of action for each class of nonnucleoside inhibitors; and (4) Quantify the effects of drug resistance mutations. In addition, hydrogen/deuterium exchange studies will reveal changes in enzyme flexibility in the transition from inactive to active enzyme, and we will attempt to determine the crystal structure of the elongation complex. This work lays the foundation for understanding structure/function relationships governing RNA-dependent RNA polymerization, the mechanisms of action of various drugs currently being investigated, and the evolution of drug resistance.