IRES-mediated translation initiation on viral mRNAs

描述(申请人提供)：许多病毒合成含有 介导帽非依赖性的内部核糖体进入位点(IRES) 翻译。IRESS彼此有很大的不同，并使用不同的机制 准备印心。我们对4-甲基-4-酮引发的分子机理的研究 不同的IRES将为理解他们的细胞提供基础 类型特异性，用于抑制剂的设计，并可识别潜在的 机械论上的相似性。IRES通常以特定于细胞类型的方式工作， 决定了病毒的致病机制。脊髓灰质炎病毒的神经毒力减弱 疫苗株是由于IRES功能缺陷，可能是由于需要 细胞特异性IRES反式作用因子(ITAFs)。我们将确定完整的 使用生化的IRES所需的一组规范因子/ITAFs 体外启动的重建，确定核糖体如何到达 起始密码子IRES和MAP下游约60个核苷酸 翻译设备的部件与IRESS的相互作用 弱毒株和强毒株。这一分析将阐明一个重要的 病毒组织嗜性的方面。甲型肝炎病毒含有580 核苷酸长的IRES也决定了病毒的生长特征和 对人类的致病性。我们将使用类似的方法来确定 本IRES和映射所需的全套规范因子和ITAFs 他们与IRES的互动。丙型肝炎病毒与板球麻痹 病毒(CRPV)IRESS使用非常不同的启动机制。丙型肝炎病毒IRES 结合eIF3和40S核糖体亚基，然后只需要eIF2/GTP/tRNA 形成一个48S复合体。我们将描述导致48秒的相互作用 详细观察复合体形成过程中eIF3‘S活性的变化规律 慢性丙型肝炎病毒感染和确定核糖体亚基如何在IRES上连接。我们 还应确定CRPV IRES如何以及在哪里结合40S亚基和 它的结合是否将eIF2/tRNA排除在P位点之外。我们将决定如何 这种IRES诱导AA-tRNA从核糖体A位移位到P位，而不需要 随之而来的多肽形成，蛋白质合成才能开始。我们会 在体外重建这一过程，以确定这一过程是如何发生的。

DESCRIPTION (provided by applicant): Many viruses synthesize mRNAs containing an internal ribosomal entry site (IRES) that mediates cap-independent translation. IRESs differ greatly from one another and use distinct mechanisms for initiation. Our studies of the molecular mechanisms of initiation on 4 different IRESs will provide a basis for understanding their cell type-specificity, for the design of inhibitors, and may identify underlying mechanistic similarities. IRESs often act in a cell type-specific manner that determines viral pathogenesis. The attenuated neurovirulence of poliovirus vaccine strains is due to defective IRES function, likely due to a need for cell-specific IRES trans-acting factors (ITAFs). We will identify the complete set of canonical factors/ITAFs required by this IRES using biochemical reconstitution of initiation in vitro, determine how ribosomes reach the initiation codon approximately 60 nucleotides downstream of the IRES and map the interactions of components of the translation apparatus with IRESs of attenuated and virulent PV strains. This analysis will elucidate an important aspect of viral tissue tropism. Hepatitis A virus contains a 580 nucleotide-long IRES that also determines viral growth characteristics and pathogenicity in humans. We shall use similar approaches to determine the complete set of canonical factors and ITAFs needed by this IRES and to map their interactions with the IRES. Hepatitis C virus (HCV) and Cricket paralysis virus (CrPV) IRESs use very different mechanisms of initiation. The HCV IRES binds both eIF3 and the 40S ribosomal subunit and then needs only eIF2/GTP/tRNA to form a 48S complex. We shall characterize interactions that lead to 48S complex formation in detail, examine regulation of eIF3's activity during chronic HCV infection and determine how ribosomal subunits join on the IRES. We shall also identify how and where the CrPV IRES binds the 40S subunit and whether its binding excludes eIF2/tRNA from the P site. We shall determine how This IRES induces translocation of aa-tRNA from ribosomal A to P site without concomitant peptide formation so that protein synthesis can begin. We shall reconstitute this process in vitro to determine how this occurs.