GENETICS OF 5' NONCODING REGIONS OF PICORNAVIRUS RNA'S

描述(申请人提供)：本提案中描述的实验将解决由微小核糖核酸病毒基因组RNA的5‘非编码区(5’NCR)编码的翻译和RNA复制功能。这些功能是蛋白质-蛋白质、RNA-蛋白质和RNA-RNA相互作用的结果，涉及病毒和细胞成分。拟议的实验将解决病毒RNA和细胞蛋白的作用，因为它们与翻译启动和RNA合成的单独活动有关。然而，由于编码I型IRES元件的微小核糖核酸病毒在帽非依赖性翻译启动过程中所利用的一些成分与参与微小核糖核酸合成的蛋白质之间存在功能重叠，因此一些拟议的实验将在这两个过程中收敛。该提案中概述的实验将定义细胞RNA结合蛋白-聚(RC)结合蛋白(PCBP2)中多聚体和/或RNA结合所需的结构域，以及这些结构域如何在感染细胞的细胞质中对病毒功能做出贡献。这些实验将解决关于PCBP2和其他细胞多肽在微小核糖核酸病毒翻译启动中的作用的重要机制问题。这项提议还将检验一种假设，该假设旨在解释微小核糖核酸病毒感染周期中同一模板(或多个模板)上假定的翻译和RNA复制之间的转换。最后，提出了体内细胞培养实验，利用最近描述的实验方法来调节细胞蛋白的蛋白水平和结构域功能，假设这些蛋白参与病毒生命周期中涉及基因组RNA5‘NCR的特定步骤。这些拟议的研究结果将为微小核糖核酸病毒翻译启动的复杂过程以及翻译功能和病毒RNA复制之间的相互作用提供新的机械性见解。此外，这些研究产生的数据应该揭示与细胞帽非依赖性翻译相关的RNA-蛋白质相互作用的基本方面，以及RNA前剪接和RNA代谢的其他元素。

DESCRIPTION (provided by applicant): The experiments described in this proposal will address the translation and RNA replication functions encoded by the 5' non-coding region (5' NCR) of picornavirus genomic RNAs. These functions are the result of protein-protein, RNA-protein, and RNA-RNA interactions involving both viral and cellular components. The proposed experiments will address the roles of viral RNAs and cellular proteins as they pertain to the separate activities of translation initiation and RNA synthesis. However, because there is functional overlap between some of the components utilized during cap-independent translation initiation by picornaviruses encoding type I IRES elements and proteins involved in picornavirus RNA synthesis, some of the proposed experiments will converge on both processes. Experiments outlined in this proposal will define the domains in the cellular RNA-binding protein, poly(rC) binding protein (PCBP2), required for multimerization and/or RNA binding and how such domains contribute to viral functions in the cytoplasm of infected cells. Such experiments will address important mechanistic questions regarding the role of PCBP2 and other cellular polypeptides in picornavirus translation initiation. This proposal will also test a hypothesis posited to explain a putative switch between translation and RNA replication on the same template (or templates) during a picornavirus infectious cycle. Finally, in vivo cell culture experiments are proposed that utilize recently described experimental approaches to modulate protein levels and domain functions of cellular proteins hypothesized to participate in specific steps in the viral life cycle that involve the genomic RNA 5' NCR. Results from these proposed studies will provide new mechanistic insights into the complex processes involved in picornavirus translation initiation and into the interplay between translation functions and viral RNA replication. In addition, data generated from such studies should reveal fundamental aspects of RNA-protein interactions relevant to cellular cap-independent translation as well as pre-mRNA splicing and other elements of RNA metabolism.