Dynamics of Hepatis C viral RNA-dependent RNA replication

描述（由申请人提供）：丙型肝炎病毒感染了大约3%的世界人口，其中包括4-5百万美国人。慢性感染导致肝硬化和癌症，2007年，丙型肝炎病毒的死亡率超过了艾滋病毒。预计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，而HCV RNA依赖性RNA聚合酶（NS5B）的抑制剂将成为该治疗的基石。FDA最近批准了一种基于首个直接抗病毒核苷类似物的新疗法，新的潜在非核苷抑制剂（NNI）目前正在研发中。这些药物是通过筛选在人肝癌细胞系中自我复制的亚基因组复制子而开发出来的。然而，由于体外RNA合成的低效率从头启动和病毒聚合酶无法从溶液中结合双链RNA（引物/模板），酶活性的生化筛选受到限制，并且普遍认为NS5B的所有晶体结构都处于失活状态。目前的酶分析呈现出一个未解决的缓慢起始动力学和快速延伸的混合物，因此，不可能知道一个给定的药物是否抑制起始或延伸。目前缺乏各类药物的结合亲和力和作用机制的定量数据。需要对酶的功能进行定量分析，以建立控制核苷酸结合、延伸和核苷酸依赖切除的动力学参数，我们最近发现这一反应可以有效地去除核苷类似物。非核苷抑制剂（NNI's）已被发现与聚合酶上至少四个不同的位点结合。这些关于各种抑制剂的数据

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.