IRES-mediated translation initiation on viral mRNAs

IRES 介导的病毒 mRNA 翻译起始

• 批准号: 6457319
• 负责人: CHRISTOPHER Ulrich Tristram HELLEN
• 金额: $ 36.92万
• 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/6457319
• 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）：许多病毒合成含有 一个内部核糖体进入位点（IRES），介导帽非依赖性 翻译.可再生能源系统彼此差异很大，使用不同的机制 入会仪式我们对4-羟基苯甲酸酯引发的分子机制进行了研究， 不同的IRES将为理解它们的细胞提供基础 类型特异性，用于抑制剂的设计，并可以识别潜在的 机械相似性IRES通常以特定于细胞类型的方式起作用， 决定了病毒的发病机制。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（ITAF）。我们将确定完整的 本IRES所需的一组典型因子/ITAF，使用生化 在体外重建起始，确定核糖体如何到达 起始密码子约60核苷酸下游的IRES和地图 翻译装置的组件与IRES的交互 减毒和毒PV菌株。这一分析将阐明一个重要的 病毒组织嗜性方面。甲型肝炎病毒含有580 也决定病毒生长特征的核苷酸长IRES， 人类的致病性。我们将使用类似的方法来确定 IRES所需的一套完整的典型因子和ITAF， 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。HCV IRES 结合eIF 3和40 S核糖体亚基，然后仅需要eIF 2/GTP/tRNA 形成48 S复合体。我们将描述导致48 S的相互作用 复合物的形成详细，检查调节eIF 3的活动， 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如何以及在何处结合40 S亚基， 它的结合是否从P位点排除eIF 2/tRNA。我们将决定如何 这种IRES诱导aa-tRNA从核糖体A位点移位到P位点， 伴随的肽形成使得蛋白质合成可以开始。我们将 在体外重建这一过程，以确定这是如何发生的。