Structure and Function of the Hepatitis C Virus Genome

丙型肝炎病毒基因组的结构和功能

• 批准号: 7148095
• 负责人: PETER SARNOW
• 金额: $ 36.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-15 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148095
• 关键词: Affect; Antiviral Therapy; Binding; Biochemical; Biochemical Genetics; Cell Line; Code; Communication; Complement; Complex; Conserved Sequence; Coupled; Data; Development; Elements; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Free Ribosome; Functional RNA; Funding; Gene Amplification; Gene Expression; Genetic; Genome; Genomics; Health; Hepatitis C; Hepatitis C virus; Indium; Initiator tRNA; Internal Ribosome Entry Site; Label; Length; Ligation; Liver; Maps; Measures; Mediating; Methods; Molecular Conformation; Monitor; Movement; Nucleotides; Outcome; Peptide Initiation Factors; Polyproteins; RNA; RNA Binding; Rate; Recruitment Activity; Regulation; Relative (related person); Replicon; Residual state; Rest; Ribosomes; Role; Scanning; Solutions; Son of Sevenless Proteins; Standards of Weights and Measures; Structure; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Transfer RNA; Translating; Translation Initiation; Translations; Viral; Viral Genome; Viral Proteins; critical developmental period; novel; novel therapeutics; research study; single molecule; single-molecule FRET; stem; viral RNA

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.

描述（由申请人提供）：丙型肝炎仍然是一个严重的健康威胁，几乎没有治疗选择。正链病毒基因组的扩增由位于病毒5'和3'非编码区的高度保守RNA元件调节。病毒蛋白的翻译由位于病毒5'非编码区的异常发散的内部核糖体进入位点（IRES）介导。IRES是一种保守的320个核苷酸的RNA，其直接结合核糖体40 S亚基以指导翻译起始。病毒负链RNA的起始受位于病毒RNA基因组3'末端的RNA茎环结构的保守景观调节。在这里，提出了一个综合的遗传，生物化学，生物物理和结构的方法来确定的机制，病毒的非编码区控制HCV的翻译和复制。在具体目标1中，生物化学和遗传学方法将描述HCV翻译起始的机制，并将揭示细胞microRNA在调节病毒RNA的基因表达中的作用。机制信息，加上大量的初步光谱数据，将用于具体目标2，以指导IRES域和全长完整IRES的NMR结构测定。为了补充静态结构实验，具体目标3使用单分子荧光光谱法探索游离和核糖体结合的IRES RNA的构象动力学，并通过直接观察翻译起始期间tRNA的结合和释放来获得机制细节。还将通过单分子荧光监测3'非编码在IRES介导的功能中的作用，以检测病毒RNA中的瞬时端到端通讯。类似地，将使用单分子荧光检查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。该提案的结果将直接影响我们对病毒复制周期中关键步骤的理解，以及新的抗病毒疗法的可能发展。