IRES-mediated translation initiation on viral mRNAs

IRES 介导的病毒 mRNA 翻译起始

• 批准号: 9107621
• 负责人: CHRISTOPHER Ulrich Tristram HELLEN
• 金额: $ 46.49万
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107621
• 关键词: Alphavirus; Animal Viruses; Antiviral Agents; Beryllium; Binding; Binding Sites; Biochemical; Brunhilde Virus; Cells; Collaborations; Complex; Consensus; Cryoelectron Microscopy; Development; Elements; Encephalomyocarditis virus; Enterovirus 71; Family Picornaviridae; Genomics; Health; Hepatitis A Virus; Human; Human Virus; Human respiratory syncytial virus; In Vitro; Indium; Individual; Initiator Codon; Initiator tRNA; Lansing Virus; Light; Link; Location; Maps; Mediating; Messenger RNA; Molecular; Molecular Conformation; Open Reading Frames; Peptide Initiation Factors; Phosphorylation; Process; Protein Biosynthesis; RNA; RNA Viruses; Reaction; Recruitment Activity; Recycling; Respiratory syncytial virus; Rhinovirus; Ribosomes; Role; Rotavirus; Signal Transduction; Sindbis Virus; Site; Staging; Structure; Testing; Translating; Translation Initiation; Translations; Viral; Viral Proteins; Virus; Virus Diseases; biophysical techniques; design; eIF-4B; inhibitor/antagonist; insight; interest; mutant; novel; pathogen; reconstitution

 DESCRIPTION (provided by applicant): Viruses depend on the host cell's translation apparatus and use diverse strategies to usurp it, to inhibit cellular translation and to evade cellular mechanisms that suppress viral translation. Some viruses disrupt the cap- binding complex eIF4F, inhibiting cap-dependent initiation, whereas eIF2 is inactivated in virus-infected host cells, impairing recruitment of initiator tRNA to the ribosome. Many viral mRNAs evade these inhibitory mechanisms by virtue of having conserved structured RNA elements that engage with components of the translation apparatus to initiate translation by non-canonical mechanisms. We will characterize three such processes - internal ribosomal entry, eIF2-independent initiation, and termination-dependent re-initiation - using in vitro reconstitution to establish factor requirements and characterize individual steps in each process. In parallel, we will use biochemical and biophysical approaches to determine the structures of novel factors, of these RNA elements, and of ribosomal initiation complexes assembled on them. Additional analysis of IRESs (internal ribosomal entry sites) will focus on the functions of conserved motifs in Type 1 (poliovirus) and Type 2 (encephalomyocarditis virus) IRESs, the mechanism of action of eIF4B and PCBP2 in initiation on them, identification of factor(s) required to expose the initiation codon sequestered at the 3'-border of the Type 1 rhinovirus IRES for ribosomal inspection, and determination of factor requirements for initiation on the structurally distinct Hepatitis A virus IRES. Analysis of eIF2-independent initiation will focus on how elements in Sindbis virus sub-genomic mRNA engage a 40S subunit so that the initiation codon is placed directly into the P- site, how Ligatin and related factors then promote binding of initiator tRNA, and whether rotavirus mRNAs use this or an unrelated mechanism to sustain translation in the absence of active eIF2. We will recapitulate termination-re-initiation on Respiratory syncytial virus M2 mRNA by in vitro reconstitution to determine which factors are required and to establish an outline mechanism for this process. Taken together, these studies will yield detailed, comprehensive insights into the interactions between viral mRNAs and components of the translation apparatus, providing a framework for understanding the mechanisms of a diversity of non-canonical initiation processes used by clinically important viruses, and for the design of inhibitors.

 描述（由申请人提供）：病毒依赖于宿主细胞的翻译装置，并使用不同的策略来篡夺它，以抑制细胞翻译并逃避抑制病毒翻译的细胞机制。一些病毒破坏帽结合复合物eIF 4F，抑制帽依赖性起始，而eIF 2在病毒感染的宿主细胞中失活，损害起始物tRNA向核糖体的募集。许多病毒mRNA通过具有保守的结构化RNA元件来逃避这些抑制机制，所述保守的结构化RNA元件与翻译装置的组分接合以通过非规范机制启动翻译。我们将表征三个这样的过程-内部核糖体进入，eIF 2独立的启动，和终止依赖性重新启动-使用体外重建，以建立因子的要求和表征每个过程中的各个步骤。与此同时，我们将使用生物化学和生物物理学的方法来确定新的因素，这些RNA元件，和核糖体起始复合物组装在它们的结构。对可再生能源战略的补充分析（内部核糖体进入位点）将集中在1型中保守基序的功能，（脊髓灰质炎病毒）和2型（脑心肌炎病毒）IRES，eIF 4 B和PCBP 2在其起始中的作用机制，鉴定暴露隔离在1型鼻病毒IRES 3 '-边界的起始密码子用于核糖体检查所需的因子，以及确定在结构上不同的甲型肝炎病毒IRES上起始所需的因子。eIF 2非依赖性起始的分析将集中在辛德毕斯病毒亚基因组mRNA中的元件如何接合40 S亚基，使得起始密码子直接置于P位点，连接蛋白和相关因子如何促进起始tRNA的结合，以及轮状病毒mRNA是否使用这种或不相关的机制在缺乏活性eIF 2的情况下维持翻译。我们将通过体外重建来概括呼吸道合胞病毒M2 mRNA的终止-重新启动，以确定需要哪些因子，并建立该过程的概要机制。总之，这些研究将产生详细的，全面的见解病毒mRNA和翻译装置的组件之间的相互作用，提供了一个框架，了解临床上重要的病毒所使用的非典型启动过程的多样性的机制，并为抑制剂的设计。