Functions of 5' NCRs of picornavirus and cellular mRNAs

描述（由申请方提供）：小核糖核酸病毒以及其他正链RNA病毒利用其基因组RNA的5'非编码区（5' NCR）中的序列结合宿主细胞和病毒蛋白，以在其细胞内复制周期中执行重要功能。脊髓灰质炎病毒、柯萨奇病毒和人鼻病毒是小核糖核酸病毒科的成员，它们在翻译起始期间进入内部核糖体所需的5'NCR中共享共同的RNA二级结构。这些结构（即，IRES元件）结合几种宿主RNA结合蛋白，包括聚（rC）结合蛋白2（PCBP 2）。PCBP 2的结合是脊髓灰质炎病毒翻译起始所必需的，这一过程也需要核质穿梭蛋白SRp 20的功能。该提案的第一个目的是确定与PCBP 2结合的脊髓灰质炎病毒IRES序列组装的翻译起始复合物的组分。遗传，生物化学和共聚焦显微镜实验将测试的假设，SRp 20作为PCBP 2结合脊髓灰质炎病毒IRES和细胞翻译起始装置的特定元素之间的分子桥梁。除了在脊髓灰质炎病毒翻译中的作用外，细胞蛋白PCBP 2也是负链病毒RNA合成所必需的。在脊髓灰质炎病毒感染HeLa细胞的过程中，PCBP 2被病毒蛋白酶切割，产生一种截短的蛋白质，该蛋白质不能在翻译中发挥作用，但保留了其在RNA复制中的作用。该提案的第二个目的将利用细胞培养和体外翻译/RNA复制方法来确定这种切割是否负责在病毒RNA合成开始之前清除脊髓灰质炎病毒基因组RNA的翻译核糖体，从而利用正链RNA模板为这两种竞争功能提供机制开关。这些研究结果将为翻译功能和小核糖核酸病毒RNA复制之间的相互作用提供新的机制见解。在该项目的一个新方向中，已经在两种细胞mRNA分子的长5'NCR中鉴定了IRES元件，一种编码电压门控钾通道（Kv1.4），另一种编码转录因子（LEF-1）。在第三个目标中提出的实验将检查与这两种细胞mRNA的5'NCR形成的RNP复合物的性质，以定义用于mRNA翻译的IRES功能的决定因素，与小核糖核酸病毒RNA不同，mRNA在细胞核中合成，并且必须在与40 S核糖体亚基缔合之前转运到细胞质。后者的研究结果将提供与小核糖核酸病毒IRES功能的重要并排比较，并对真核细胞如何启动具有长的高度结构化5'NCR的mRNA的翻译提供新的见解。公共卫生相关性：本计画将探讨人类微小核糖核酸病毒感染细胞后，其基因表现及病毒RNA复制之机制。它还将分析一类特殊的哺乳动物细胞蛋白质合成，在某些生理和细胞特异性条件下是重要的。这项研究将增强我们对病毒-宿主相互作用以及基因表达的正常细胞机制的认识。

DESCRIPTION (provided by applicant): Picornaviruses as well as other positive strand RNA viruses utilize sequences in the 5' noncoding regions (5' NCRs) of their genomic RNAs to bind host cell and viral proteins to carry out important functions during their intracellular replication cycles. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA secondary structure in their 5' NCRs required for internal ribosome entry during translation initiation. These structures (i.e., IRES elements) bind several host RNA binding proteins, including poly(rC) binding protein 2 (PCBP2). Binding of PCBP2 is required for poliovirus translation initiation, a process that also requires the function of a nucleo-cytoplasmic shuttling protein, SRp20. The first aim of this proposal is to identify components of translation initiation complexes assembled with PCBP2-bound poliovirus IRES sequences. Genetic, biochemical, and confocal microscopy experiments will test the hypothesis that SRp20 acts as a molecular bridge between PCBP2 bound to the poliovirus IRES and specific elements of the cellular translation initiation apparatus. In addition to its role in poliovirus translation, cellular protein PCBP2 is required for negative-strand viral RNA synthesis. During poliovirus infection of HeLa cells, PCBP2 is cleaved by a viral proteinase to generate a truncated protein that is unable to function in translation but retains its role in RNA replication. The second aim of the proposal will utilize cell culture and in vitro translation/RNA replication approaches to determine if this cleavage is responsible for clearing poliovirus genomic RNAs of translating ribosomes prior to the onset of viral RNA synthesis, providing a mechanistic switch for these two competing functions utilizing positive- strand RNA templates. Results from these proposed studies will provide new mechanistic insights into the interplay between translation functions and picornavirus RNA replication. In a new direction for this project, IRES elements have been identified in the long 5' NCRs of two cellular mRNA molecules, one that encodes a voltage-gated potassium channel (Kv1.4) and one that encodes a transcription factor (LEF-1). The experiments proposed in the third aim will examine the nature of RNP complexes formed with the 5' NCRs of these two cellular mRNAs to define the determinants of IRES functions for translation of mRNAs that, unlike picornavirus RNAs, are synthesized in the nucleus of cells and must be transported to the cytoplasm prior to their association with 40S ribosomal subunits. Results from these latter studies will provide important side-by-side comparisons with picornavirus IRES functions and new insights into how eukaryotic cells initiate translation from mRNAs with long, highly-structured 5' NCRs. PUBLIC HEALTH RELEVANCE: This project will elucidate the mechanisms of gene expression and viral RNA replication in cells infected by human picornaviruses. It will also analyze a specialized class of mammalian cell protein synthesis that is important under certain physiological and cell- specific conditions. The proposed research will enhance our knowledge of virus-host interactions as well as normal cell mechanisms of gene expression.