INITIATION OF HEPATITIS C VIRUS TRANSLATION

丙型肝炎病毒（HCV）基因组的翻译由大约350 nt介导。长内部核糖体进入位点（IRES）。核糖体亚基（40 S）与HCV IRES形成稳定的二元复合物。 这种RNA的复杂结构决定了起始密码子在这些复合物中核糖体“P”位点的正确定位。 Met-tRNA、eIF 2和GTP加入到二元复合物中导致形成48 S预起始复合物。本提案的目的是表征IRES和细胞翻译装置的组件在启动过程中如何相互作用，以阐明HCV在分子水平启动的独特机制，并确定IRES中抑制其功能的化疗药物的潜在靶点。UV交联、化学足迹和突变分析将用于鉴定（i）IRES与40 S亚基相互作用的决定因素和（ii）构成该亚基上IRES结合位点的核糖体蛋白和核糖体RNA片段。 然后将结合决定簇与结合位点匹配。 相同的方法将用于分析当eIF 2、起始剂tRNA和GTP与二元核糖体复合物结合形成48 S预起始复合物时发生的IRES的构象重排。 eIF 3（与IRES特异性结合）在此阶段不需要，但对于随后连接60 S亚基形成活性80 S核糖体复合物至关重要。 将确定eIF 3与IRES相互作用的决定因素，然后在纯化组分体外重构的起始反应中评估其对IRES介导的起始中eIF 3活性的重要性。

Translation of the hepatitis C virus (HCV) genome is mediated by a approximately 350nt.-long internal ribosomal entry site (IRES). Ribosomal subunits (40S) form stable binary complexes with the HCV IRES. The complex structure of this RNA determines the correct positioning of the initiation codon in the ribosomal 'P' site in these complexes. Addition of Met-tRNA, elF2 and GTP to binary complexes results in formation of 48S preinitiation complexes. The aim of this proposal is to characterize how the IRES and components of the cellular translation apparatus interact during initiation, in order to elucidate the unique mechanism of HCV initiation at the molecular level and to identify potential targets in this IRES for chemotherapeutic agents that will inhibit its function. UV cross-linking, chemical footprinting and mutational analyses will be used to identify (i) determinants in the IRES of its interaction with the 40S subunit and (ii) ribosomal proteins and segments of ribosomal RNA that constitute IRES-binding sites on this subunit. Binding determinants will then be matched with binding sites. The same approaches will be used to analyse the conformational rearrangement of the IRES that occurs when eIF2, initiator tRNA and GTP bind to the binary ribosomal complex to form a 48S preinitiation complex. eIF3 (which binds specifically to the IRES) is not required at this stage but is essential for subsequent joining of the 60S subunit to form an active 80S ribosomal complex. Determinants of eIF3's interaction with the IRES will be identified and their importance for eIF3's activity in IRES-mediated initiation will then be assessed in initiation reactions reconstituted in vitro from purified components.