Functions of 5' NCRs of picornavirus and cellular mRNAs

描述（由申请人提供）：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’ NCRs）中的序列结合宿主细胞和病毒蛋白，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，它们在翻译起始过程中进入内部核糖体所需的5' ncr中具有共同的RNA二级结构。这些结构（即IRES元件）结合多种宿主RNA结合蛋白，包括聚（rC）结合蛋白2 （PCBP2）。脊髓灰质炎病毒翻译起始需要PCBP2的结合，这一过程也需要核胞质穿梭蛋白SRp20的功能。本提案的第一个目的是鉴定与pcbp2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、生化和共聚焦显微镜实验将验证SRp20作为与脊髓灰质炎病毒IRES结合的PCBP2和细胞翻译起始装置特定元件之间的分子桥梁的假设。除了在脊髓灰质炎病毒翻译中发挥作用外，细胞蛋白PCBP2也是负链病毒RNA合成所必需的。在脊髓灰质炎病毒感染HeLa细胞期间，PCBP2被病毒蛋白酶切割，产生一个截断的蛋白，该蛋白不能在翻译中起作用，但保留其在RNA复制中的作用。该提案的第二个目标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合成开始之前清除翻译核糖体的脊髓灰质炎病毒基因组RNA，并利用正链RNA模板为这两种竞争功能提供机制开关。这些研究的结果将为翻译功能与小核糖核酸复制之间的相互作用提供新的机制见解。在这个项目的一个新方向，IRES元件已经在两个细胞mRNA分子的长5' ncr中被鉴定出来，一个编码电压门控钾通道（Kv1.4），一个编码转录因子（LEF-1）。第三个目标中提出的实验将检查与这两种细胞mrna的5' NCRs形成的RNP复合物的性质，以确定IRES翻译mrna功能的决定因素。与小核糖核酸rna不同，mrna是在细胞核中合成的，必须在与40S核糖体亚基结合之前转运到细胞质中。后一项研究的结果将提供与小核糖核酸病毒IRES功能的重要并排比较，并为真核细胞如何启动具有长且高度结构化的5' NCRs的mrna的翻译提供新的见解。公共卫生相关性：本项目将阐明人小核糖核酸病毒感染细胞中基因表达和病毒RNA复制的机制。它还将分析一类特殊的哺乳动物细胞蛋白质合成，这在某些生理和细胞特异性条件下是重要的。本研究将增强我们对病毒与宿主相互作用以及正常细胞基因表达机制的认识。

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.