Dynamics of Hepatis C viral RNA-dependent RNA replication

描述(申请人提供)：丙型肝炎病毒感染了大约3%的世界人口，其中包括美国的400万至500万人。慢性感染会导致肝硬变和癌症，在2007年，丙型肝炎病毒的死亡率超过了艾滋病毒。预计成功治疗丙型肝炎病毒感染将需要类似于目前治疗艾滋病毒感染的综合疗法，而丙型肝炎病毒RNA依赖的RNA聚合酶(NS5B)的抑制剂将是该疗法的基石。FDA最近批准了一种基于第一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。这些药物是通过使用基于亚基因组复制子的筛选来开发的，这些复制子在人类肝癌细胞系中自我复制。然而，由于体外RNA合成从头开始的低效以及病毒聚合酶不能从溶液中结合双链RNA(引物/模板)，用于酶活性的生化筛选一直是有限的，而且人们普遍认为NS5B的所有晶体结构都处于非活性状态。目前的酶分析呈现出缓慢的起始动力学和快速的延伸的未解决的混合物，因此，不可能知道特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。需要一种酶功能的定量测定来建立控制核苷酸掺入、延伸和核苷酸依赖的切除的动力学参数，我们最近证明这种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地形成和纯化活性的、高度进行性的延长复合体的条件，克服了对NS5B催化的复制进行详细的生化分析的主要障碍。在这个提案中，我们将使用最先进的单核苷酸转换动力学方法：(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)检查核苷酸类似物的掺入、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；以及(4)量化耐药突变的影响。此外，氢/氚交换研究将揭示酶从失活到活性转变过程中柔韧性的变化，我们将试图确定延长复合体的晶体结构。这项工作为理解RNA依赖的RNA聚合的结构/功能关系，目前正在研究的各种药物的作用机制，以及耐药性的演变奠定了基础。

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.