IRES -mediated translation initiation on viral mRNAs

许多正义RNA病毒的基因组包含一个内部核糖体进入位点(IRES)，该位点 调解独立于终端的翻译启动。IRESS属于不同的结构类，并使用 不同的启动机制。我们建议对代表的启动过程进行研究 使用日益复杂的机制的四类IRES将识别重要的顺式作用 元素，并提供对规范启动因子和 促进核糖体内部进入的细胞IRES反式作用因子(ITAFs)。我们的研究将解决 增加复杂性的机制。~180nt的基因组间隔区(IGR)序列 双胞菌病毒在没有启动子tRNA或启动因子的情况下介导启动和第一个延伸周期 因此，在核糖体P位点上没有脱酰tRNA。我们将确定是否 IRES中的元件在延伸过程中起着类似于脱酰化tRNA的作用 被转移到E站点。猪瘟病毒的IRES具有很强的抗药性 对eIF2磷酸化的抑制，这可以通过使用两个例外的eIF2- 独立的启动机制。我们将描述这些机制是否解释了 在核糖体的这个IRES上的松弛？S通常严格避免在非AUG密码子上启动。类型 2微小核糖核酸病毒IRES如脑心肌炎病毒IRES与eIF4G/eIF4A特异性结合：我们将 研究这些因素如何促进43S复合体招募到起始密码子，如何 ITAFs促进eIF4G/eIF4A的结合，以及为什么只有IRESS类型的子集需要它们的活动。这个 I型小核糖核酸病毒IRESS的启动机制，如脊髓灰质炎病毒，尚不清楚。我们会 通过提纯和识别所有必要的因素来表征它，以便重建整个提升体 在体外过程中。然后我们将用以下方法描述因子和核糖体之间的相互作用 化学/酶足迹和定向羟基自由基裂解。这些研究将提供一个 了解IRES介导的启动的机制细节的框架，用于了解 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.