IRES-mediated translation initiation on viral mRNAs

IRES 介导的病毒 mRNA 翻译起始

• 批准号: 6622818
• 负责人: CHRISTOPHER Ulrich Tristram HELLEN
• 金额: $ 36.93万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622818
• 关键词: Hepatovirus; Picornaviridae; cryoelectron microscopy; genetic mapping; genetic translation; hepatitis C virus; messenger RNA; nucleic acid sequence; poliovirus; transcription factor; virus RNA; virus genetics

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.

描述（由申请人提供）：许多病毒合成的 mRNA 含有 介导帽独立的内部核糖体进入位点 (IRES) 翻译。 IRES 彼此之间差异很大，并且使用不同的机制 用于启动。我们对 4 起始分子机制的研究 不同的 IRES 将为了解其细胞提供基础 类型特异性，用于抑制剂的设计，并且可以识别潜在的 机制上的相似性。 IRES 通常以细胞类型特异性的方式起作用， 决定病毒的发病机制。脊髓灰质炎病毒的神经毒力减弱 疫苗株的 IRES 功能缺陷，可能是由于需要 细胞特异性 IRES 反式作用因子 (ITAF)。我们将确定完整的 本 IRES 使用生化所需的一组规范因素/ITAF 体外重构起始，确定核糖体如何到达 起始密码子位于 IRES 和图谱下游约 60 个核苷酸处 翻译装置的组件与 IRES 的相互作用 减毒和强毒 PV 菌株。这一分析将阐明一个重要的 病毒组织趋向性方面。第580章 甲型肝炎病毒 长核苷酸 IRES 也决定病毒生长特征 对人类有致病性。我们将使用类似的方法来确定 该 IRES 所需的完整规范因素和 ITAF 集并映射 他们与 IRES 的互动。丙型肝炎病毒 (HCV) 和蟋蟀麻痹 病毒 (CrPV) IRES 使用非常不同的启动机制。丙肝病毒 IRES 同时结合 eIF3 和 40S 核糖体亚基，然后仅需要 eIF2/GTP/tRNA 形成48S复合体。我们将描述导致 48S 的交互作用 详细了解复合物的形成，检查 eIF3 活性的调节 慢性 HCV 感染并确定核糖体亚基如何加入 IRES。我们 还应确定 CrPV IRES 结合 40S 亚基的方式和位置，以及 其结合是否将 eIF2/tRNA 从 P 位点排除。我们将决定如何 该 IRES 诱导 aa-tRNA 从核糖体 A 易位至 P 位点，而无需 伴随肽形成，从而开始蛋白质合成。我们将 在体外重建这个过程以确定这是如何发生的。