Structure and Function of the Hepatitis C Virus Genome

描述（由申请人提供）：丙型肝炎仍然是一种严重的健康威胁，治疗选择很少。正链病毒基因组的扩增是由位于病毒5′和3′非编码区高度保守的RNA元件调控的。病毒蛋白的翻译是由位于病毒5'非编码区异常发散的内部核糖体进入位点（IRES）介导的。IRES是一种保守的320核苷酸RNA，直接结合核糖体40S亚基直接翻译起始。病毒负链RNA的起始是由位于病毒RNA基因组非常3'端的RNA茎环结构的保守景观调节的。本文提出了一种结合遗传、生化、生物物理和结构的方法来确定病毒非编码区控制HCV翻译和复制的机制。在特定的目标1中，生化和遗传方法将描述HCV翻译起始的机制，并将揭示细胞microRNA在调节病毒RNA基因表达中的作用。在具体目标2中，将利用这些机制信息，结合大量的初步光谱数据，指导IRES结构域和全长完整IRES的核磁共振结构确定。为了补充静态结构实验，具体目标3利用单分子荧光光谱技术探索游离和核糖体结合的IRES RNA的构象动力学，并通过直接观察tRNA在翻译起始过程中的结合和释放来获得机制细节。3'非编码在ires介导功能中的作用也将通过单分子荧光检测病毒RNA中的瞬时端到端通信。同样，microrna -病毒RNA相互作用对位于病毒基因组3'端的保守序列元件的影响将使用单分子荧光进行检测。这一建议的结果将直接影响我们对病毒复制周期关键步骤的理解，并可能开发新的抗病毒疗法。

DESCRIPTION (provided by applicant): Hepatitis C remains a serious health threat, with few therapeutic options. Amplification of the positive-stranded viral genome is regulated by highly conserved RNA elements located in the viral 5' and 3' noncoding regions. Translation of the viral proteins is mediated by an unusually divergent internal ribosome entry site (IRES) located in the viral 5' noncoding region. The IRES is a conserved 320 nucleotide RNA that binds directly to ribosomal 40S subunits to direct translation initiation. Initiation of viral minus-stranded RNAs is modulated by a conserved landscape of RNA stem-loop structures located at the very 3' end of the viral RNA genome. Here, a combined genetic, biochemical, biophysical and structural approach is proposed to determine the mechanism by which the viral noncoding regions control HCV translation and replication. In specific aim 1, biochemical and genetic methods will delineate the mechanisms of HCV translation initiation, and will unravel the roles of a cellular microRNA in modulating gene expression of the viral RNA. The mechanistic information, coupled with a large amount of preliminary spectroscopic data, will be used in specific aim 2 to guide NMR structure determinations of IRES domains and the full-length intact IRES. To complement static structural experiments, specific aim 3 explores the conformational dynamics of free and ribosome-bound IRES RNA using single-molecule fluorescence spectroscopy, and mechanistic details will be obtained by direct observation of tRNA binding and release during translation initiation. The role of the 3' noncoding in IRES-mediated function will be also monitored by single-molecule fluorescence to detect transient end-to-end communication in viral RNA. Similarly, the effects of microRNA-viral RNA interactions on conserved sequence elements located at the 3' end of the viral genome will be examined using single-molecule fluorescence. The results of this proposal will have direct impact on our understanding of critical steps in the viral replication cycle, and the possible development of novel antiviral therapies.