IRES-mediated translation initiation on viral mRNAs

描述（由申请方提供）：许多正义RNA病毒的基因组含有介导非末端依赖性翻译起始的内部核糖体进入位点（IRES）。IRES属于不同的结构类别，并使用不同的启动机制。我们提出的四类IRES的代表，使用不同的机制的启动过程的研究将确定重要的顺式作用元件，并提供详细的机制的见解典型的启动因子和细胞IRES反式作用因子（ITAFs）在促进内部核糖体进入的行动。我们的研究将解决日益复杂的机制。双顺反子病毒的~180 nt长的基因组间区域（IGR）IRES在没有起始剂tRNA或起始因子的情况下介导起始，因此在核糖体P位点没有脱酰化的tRNA的情况下发生第一个延伸循环。我们将表征IRES中的元件是否在脱酰tRNA的延伸过程中发挥类似的作用，当它被易位到E位点时。经典猪瘟病毒（一种瘟病毒）的IRES对eIF 2磷酸化的抑制具有实质性抗性，这可以通过使用两种特殊的不依赖于eIF 2的起始机制来解释。我们将确定这些机制是否解释了核糖体通常严格避免在非AUG密码子处起始的IRES上的松弛。2型小核糖核酸病毒IRES如脑心肌炎病毒IRES特异性结合eIF 4G/eIF 4A：我们将研究这些因子如何促进43 S复合物募集到起始密码子，ITAFs如何诱导这些IRES的构象变化，以及为什么只有一部分2型IRES需要它们的活性。1型小核糖核酸病毒（例如脊髓灰质炎病毒）的起始机制尚不清楚，但也涉及与eIF 4G的特异性相互作用。我们将详细描述这种相互作用，验证先前确定的候选ITAF，并在必要时分离额外的ITAF，以在体外重建该IRES的整个启动过程。然后，我们将使用化学/酶足迹法和定向羟基自由基裂解来表征因子和IRES之间的相互作用。这些研究将为理解IRES介导的启动机制细节、理解IRES功能的细胞类型特异性和设计抑制剂提供框架。

DESCRIPTION (provided by applicant): 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 distinct mechanisms 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 ~180 nt-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 in the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of classical swine fever virus, a pestivirus, 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 determine whether these mechanisms account for the relaxation on this IRES of the ribosome's normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such as the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors promote recruitment of 43S complexes to the initiation codon, how ITAFs induce conformational changes in these IRESs and why only a subset of type 2 IRESs require their activity. The mechanism of initiation on type 1 picornaviruses (e.g. poliovirus) is not known, but also involves specific interaction with eIF4G. We shall characterize this interaction in detail, validate previously identified candidate ITAFs and if necessary isolate additional ITAFs to reconstitute the entire initiation process on this IRES in vitro. We will then characterize interactions between factors and the IRES 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.