INITIATION OF HEPATITIS C VIRUS TRANSLATION

丙型肝炎病毒(丙型肝炎病毒)基因组的翻译是由大约350个核苷酸的内部核糖体进入位点(IRES)介导的。核糖体亚基(40S)与丙型肝炎病毒IRES形成稳定的二元复合体。这种RNA的复杂结构决定了起始密码子在这些复合体的核糖体‘P’位点上的正确定位。将Met-tRNA、elF2和GTP加入到二元络合物中，形成48S预引发络合物。本研究的目的是为了从分子水平上阐明丙型肝炎病毒启动的独特机制，并找出抑制其功能的化疗药物的潜在靶点。将使用紫外光交联、化学足迹和突变分析来确定(I)其与40S亚基相互作用的IRES中的决定因素，以及(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，结合决定簇将与结合位点相匹配。同样的方法将被用于分析当eIF2、启动子tRNA和GTP与二元核糖体复合体结合形成48S预引发复合体时发生的IRES的构象重排。EIF3(它与IRES特异结合)在这个阶段不是必需的，但对于随后60S亚单位的加入形成活性的80S核糖体复合体是必不可少的。我们将确定eIF3‘S与IRES相互作用的决定因素，并将在由纯化成分在体外重组的引发反应中评估它们对eif3’S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.