Functions of 5' NCRs of picornavirus and cellular mRNAs

描述(申请人提供)：微小核糖核酸病毒和其他正链RNA病毒利用其基因组RNA 5‘非编码区(5’NCR)的序列与宿主细胞和病毒蛋白结合，在其细胞内复制周期中执行重要功能。脊髓灰质炎病毒、柯萨奇病毒和人类鼻病毒都是短小核糖科的成员，它们的5‘NCR具有共同的RNA二级结构，在翻译启动过程中需要内部核糖体进入。这些结构(即IRES元件)与几种宿主RNA结合蛋白结合，包括聚(RC)结合蛋白2(PCBP2)。结合PCBP2是启动脊髓灰质炎病毒翻译所必需的，这一过程也需要核质穿梭蛋白SRp20的功能。这项建议的第一个目标是确定与PCBP2结合的脊髓灰质炎病毒IRES序列组装的翻译起始复合体的组成部分。遗传、生化和共聚焦显微镜实验将检验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的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的作用外，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，产生一种截短的蛋白质，该蛋白质无法在翻译中发挥功能，但保留了其在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合成开始之前清除脊髓灰质炎病毒基因组RNA的翻译核糖体，利用正链RNA模板为这两种相互竞争的功能提供机制开关。这些拟议研究的结果将为翻译功能和小核糖核酸复制之间的相互作用提供新的机械性见解。在这个项目的新方向上，已经在两个细胞mRNA分子的长5‘NCR中发现了IRES元件，一个编码电压门控钾通道(Kv1.4)，另一个编码转录因子(Lef-1)。第三个目标中提出的实验将研究这两个细胞mRNA5‘NCRs形成的RNP复合体的性质，以确定mRNAs翻译的IRES功能的决定因素，与小核糖核酸不同，RNP在细胞核中合成，在与40S核糖体亚基结合之前必须运输到细胞质。后一项研究的结果将提供与微小核糖核酸病毒IRES功能的重要并列比较，并为真核细胞如何从具有长的、高结构的5‘NCRs的mRNAs启动翻译提供新的见解。公共卫生相关性：该项目将阐明感染人类微小核糖核酸病毒的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有的条件下是重要的。这项拟议的研究将加强我们对病毒-宿主相互作用以及正常细胞基因表达机制的了解。

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.