IRES -mediated translation initiation on viral mRNAs

许多正义RNA病毒的基因组含有内部核糖体进入位点（IRES）， 介导末端独立的翻译起始。IRES属于不同的结构类，并使用 不同的启动机制。我们提出的研究过程中的启动代表 四类使用日益复杂的机制的IRES将确定重要的顺式作用 元素，并提供了详细的机械见解的行动，典型的启动因素， 细胞IRES反式作用因子（ITAFs）促进内部核糖体进入。我们的研究将解决 日益复杂的机制。用~ 180 nt的IGR序列分析法， 双顺反子病毒在没有起始tRNA或起始因子的情况下介导起始， 因此，在核糖体P位点没有脱酰tRNA的情况下发生。我们将描述 IRES中的元件在延伸到脱酰化的tRNA时起类似的作用， 转移到E位点。猪瘟病毒的IRES具有实质性耐药性 通过eIF 2磷酸化抑制，这可以通过使用两种特殊的eIF 2- 独立的启动机制。我们将描述这些机制是否解释了 核糖体的IRES上的松弛通常严格避免在非AUG密码子处起始。类型 2种小核糖核酸病毒IRES，如脑心肌炎病毒IRES，特异性结合eIF 4G/eIF 4A：我们将 研究这些因素如何促进43 S复合物募集到起始密码子， ITAF促进eIF 4G/eIF 4A的结合，以及为什么只有IRES类型的子集需要它们的活性。的 1型小核糖核酸病毒IRES（如脊髓灰质炎病毒）的起始机制尚不清楚。我们将 通过净化和识别所有必要的因素来表征它，以重建整个启动 体外过程。然后，我们将使用以下方法描述因子和核糖体之间的相互作用： 化学/酶足迹法和定向羟基自由基裂解。这些研究将提供一个 理解IRES介导的启动机制细节的框架， IRES功能的细胞类型特异性和抑制剂的设计。

The genomes of many positive sense RNA viruses contain an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation. IRESs belong to different structural classes and use distinct mechanisms for initiation. Our proposed studies of the process of initiation on representatives of four classes of IRES that use mechanisms of increasing complexity will identify important cis-acting elements and provide detailed mechanistic insights into the actions of canonical initiation factors and cellular IRES trans-acting factors (ITAFs) in promoting internal ribosomal entry. Our studies will address mechanisms of increasing complexity. The ~180nt-long intergenomic region (IGR) IRESs of dicistroviruses mediate initiation without initiator tRNA or initiation factors and the first elongation cycle consequently occurs without deacylated tRNA in the ribosomal P site. We will characterize whether elements within the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of the pestivirus classical swine fever virus is substantially resistant to inhibition by eIF2 phosphorylation, which can be accounted for by the use of two exceptional eIF2- independent mechanisms of initiation. We will characterize whether these mechanisms account for relaxation on this IRES of the ribosome¿s normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors act to promote recruitment of 43S complexes to the initiation codon, how ITAFs promote binding of eIF4G/eIF4A and why only a subset of type IRESs require their activity. The mechanism of initiation on type 1 picornavirus IRESs, such as that of poliovirus, is not known. We shall characterize it by purifying and identifying all necessary factors in order to reconstitute the entire initiation process in vitro. We will then characterize interactions between factors and the ribosome using chemical/enzymatic footprinting and directed hydroxyl radical cleavage. The studies will provide a framework for understanding of mechanistic details of IRES-mediated initiation, for understanding the cell-type specificity of IRES function and for the design of inhibitors.