IRES-mediated translation initiation on viral mRNAs

 描述(申请人提供)：病毒依赖于宿主细胞的翻译装置，并使用不同的策略来篡夺它，抑制细胞翻译，并逃避抑制病毒翻译的细胞机制。一些病毒破坏帽结合复合体eIF4F，抑制帽依赖的启动，而eIF2在病毒感染的宿主细胞中失活，损害启动子tRNA到核糖体的募集。许多病毒mRNAs通过保守的结构RNA元件与翻译装置的组件相结合来通过非规范机制启动翻译来逃避这些抑制机制。我们将描述三个这样的过程-内部核糖体进入，eIF2独立的启动，和终止依赖的重新启动-使用体外重组来确定因子要求，并表征每个过程中的个别步骤。同时，我们将使用生化和生物物理方法来确定新因子的结构，这些RNA元件以及组装在它们上面的核糖体起始复合体的结构。对IRESS(内部核糖体进入位点)的进一步分析将集中在1型(脊髓灰质炎病毒)和2型(脑心肌炎病毒)IRESS中保守基序的功能，eIF4B和PCBP2在它们启动过程中的作用机制，揭示隔离在I型鼻病毒IRES 3‘端边界的启动密码子所需的因子(S)的鉴定，以及在结构上不同的甲型肝炎病毒IRES上启动所需的因子的确定。对eIF2非依赖性启动的分析将集中在Sindbis病毒亚基因组mRNA中的元件如何与40S亚基结合，从而将起始密码子直接放置到P-位点，连接蛋白和相关因素如何促进启动子tRNA的结合，以及轮状病毒mRNAs是否使用这种机制或在没有活性eIF2的情况下使用无关的机制来维持翻译。我们将通过体外重组概述呼吸道合胞病毒M2基因的终止-再启动过程，以确定需要哪些因素并建立这一过程的基本机制。综上所述，这些研究将对病毒mRNAs和翻译装置组件之间的相互作用产生详细、全面的见解，为理解临床上重要病毒使用的各种非规范启动过程的机制和抑制剂的设计提供一个框架。

 DESCRIPTION (provided by applicant): Viruses depend on the host cell's translation apparatus and use diverse strategies to usurp it, to inhibit cellular translation and to evade cellular mechanisms that suppress viral translation. Some viruses disrupt the cap- binding complex eIF4F, inhibiting cap-dependent initiation, whereas eIF2 is inactivated in virus-infected host cells, impairing recruitment of initiator tRNA to the ribosome. Many viral mRNAs evade these inhibitory mechanisms by virtue of having conserved structured RNA elements that engage with components of the translation apparatus to initiate translation by non-canonical mechanisms. We will characterize three such processes - internal ribosomal entry, eIF2-independent initiation, and termination-dependent re-initiation - using in vitro reconstitution to establish factor requirements and characterize individual steps in each process. In parallel, we will use biochemical and biophysical approaches to determine the structures of novel factors, of these RNA elements, and of ribosomal initiation complexes assembled on them. Additional analysis of IRESs (internal ribosomal entry sites) will focus on the functions of conserved motifs in Type 1 (poliovirus) and Type 2 (encephalomyocarditis virus) IRESs, the mechanism of action of eIF4B and PCBP2 in initiation on them, identification of factor(s) required to expose the initiation codon sequestered at the 3'-border of the Type 1 rhinovirus IRES for ribosomal inspection, and determination of factor requirements for initiation on the structurally distinct Hepatitis A virus IRES. Analysis of eIF2-independent initiation will focus on how elements in Sindbis virus sub-genomic mRNA engage a 40S subunit so that the initiation codon is placed directly into the P- site, how Ligatin and related factors then promote binding of initiator tRNA, and whether rotavirus mRNAs use this or an unrelated mechanism to sustain translation in the absence of active eIF2. We will recapitulate termination-re-initiation on Respiratory syncytial virus M2 mRNA by in vitro reconstitution to determine which factors are required and to establish an outline mechanism for this process. Taken together, these studies will yield detailed, comprehensive insights into the interactions between viral mRNAs and components of the translation apparatus, providing a framework for understanding the mechanisms of a diversity of non-canonical initiation processes used by clinically important viruses, and for the design of inhibitors.