Defining the Structural Mechanisms of RNP Complexes that Regulate Enterovirus Translation

定义调节肠道病毒翻译的 RNP 复合物的结构机制

• 批准号: 10092187
• 负责人: Gary A. Brewer
• 金额: $ 35.99万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092187
• 关键词: 5' Untranslated Regions; AUF1A protein; Adopted; Antiviral Agents; Base Pairing; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Biophysics; Cells; Cellular Assay; Cessation of life; Chemicals; Cleaved cell; Complex; Data; Dicer Enzyme; Disease Outbreaks; Elements; Ensure; Enterovirus; Enterovirus 71; Event; Foundations; Gene Expression; Genes; Genetic; Genome; Goals; Impairment; Infection; Integration Host Factors; Internal Ribosome Entry Site; Knowledge; Lead; Mission; Modeling; Molecular; Molecular Conformation; Morbidity - disease rate; Mutation; National Institute of General Medical Sciences; Pathogenesis; Pathogenicity; Phylogenetic Analysis; Positioning Attribute; Protein Biosynthesis; Protein Conformation; Proteins; Public Health; RNA; RNA Conformation; RNA-Binding Proteins; Regulation; Research; Ribonucleoproteins; Ribosomes; Series; Small RNA; Specificity; Structural Biochemistry; Structure; Testing; Translations; United States National Institutes of Health; Vaccines; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Replication; Work; advanced disease; effective therapy; emerging pathogen; global health; hnRNP A1; hnRNP protein A1; innovation; insight; nervous system disorder; novel; programs; recruit; siRNA delivery; stem; success; viral RNA; virology

Project abstract: Enteroviruses (EVs) coordinate protein synthesis and genome replication through their 5'UTR, which is predicted to fold into six stem loops. Stem loop (SL) I facilitates viral genome replication; SLII- VI function as the basic IRES unit for cap-independent translation. Several host RNA binding proteins (referred to as ITAFs), are recruited to the IRES for efficient viral protein synthesis. The current dogma supports a model wherein ITAFs either stabilize or destabilize IRES structure in a conformation that controls ribosome entry. The fundamental knowledge gap is an understanding of mechanisms by which ITAFs functionally interact with the 5'UTR to organize IRES structure. This knowledge is critical to reveal underlying mechanisms by which EVs redirect host factors to maintain infection. The long-term goal is to better understand the molecular mecha- nisms by which EVs subvert host factors to regulate viral gene expression and replication. The overall objec- tive of this proposal is to illuminate how ITAFs functionally interact with the viral IRES to control translation. EV71 and the ITAFs hnRNP A1 and AUF1 serve as excellent models as they bind the same IRES domain SLII to antagonistically fine-tune EV71 translation levels. Moreover, vsRNA1, a virus-derived, small RNA produced by Dicer cleavage of the SLII IRES region, further represses EV71 translation. Our central hypothesis is that conserved viral RNA elements fine-tune EV71 translation levels by assembling unique and antagonistic ribo- nucleoprotein (RNP) complexes, and vsRNA1 compete these interactions to permit more stringent control. The rationale for this research is that ITAF-IRES interactions are central to the synthesis of every EV71 protein and its replication. This new knowledge will thus prove applicable to similar EVs as well. Strong preliminary data lead to three specific aims: (1) Determine the structures of IRES elements that contribute to viral replication; (2) reveal the detailed molecular interactions of antagonistic ITAFs that coordinate viral translation; and (3) identify mechanisms by which viral small RNAs inhibit translation. For Aim1, phylogenetics, chemical probing, NMR, SAXS, and cellular assays will define IRES structural elements that contribute to EV71 translation. For Aim 2, structural, biophysical, and virological assays will reveal protein and RNA conformational changes, and func- tional changes, upon binding of hnRNP A1 and AUF1 to the IRES SLII region, which is critical to translational control. For Aim 3, structural, biochemical, and virological assays will reveal the structural and functional ef- fects of vsRNA1 on antagonistic hnRNP A1- and AUF1-RNPs. The proposal is innovative by employing an in- tegrated program combining structural biochemistry, biophysics, and virological studies that promise to provide significant breakthroughs to better understand viral-host interactions that contribute to EV71 pathogenesis. The research is significant since it promises to deliver unprecedented insights into RNPs and RNA-RNA interac- tions that contribute to EV pathogenesis. This will in turn provide a foundation for identification of new antivirals against EV71 and EV68, which the NIH has recently designated as emerging pathogens.

项目摘要：肠道病毒（EV）通过其蛋白质合成和基因组复制来协调蛋白质合成和基因组复制。 5 'UTR，其被预测折叠成六个茎环。茎环（SL）I促进病毒基因组复制; SLII- VI作为IRES的基本单元，用于cap-independent翻译。几种宿主RNA结合蛋白（参见 称为ITAF）被募集到IRES用于有效的病毒蛋白合成。目前的教条支持一种模式， 其中ITAF使IRES结构在控制核糖体进入的构象中稳定或不稳定。的 一个基本的知识差距是对ITAF在功能上与 5 'UTR组织IRES结构。这一知识对于揭示EV的潜在机制至关重要。 重定向宿主因子以维持感染。长期目标是更好地理解分子机制- EV通过其破坏宿主因子以调节病毒基因表达和复制。总体目标-- 这个提议的目的是阐明ITAF如何在功能上与病毒IRES相互作用以控制翻译。 EV 71和ITAF hnRNP A1和AUF 1作为极好的模型，因为它们结合相同的IRES结构域SLII 对抗性地微调EV 71的翻译水平。此外，vsRNA 1是一种病毒衍生的小RNA， 通过SLII IRES区域的Dicer切割，进一步抑制EV 71翻译。我们的核心假设是， 保守的病毒RNA元件通过组装独特的和拮抗性的核糖核酸来微调EV 71的翻译水平， 核蛋白（RNP）复合物和vsRNA 1竞争这些相互作用，以允许更严格的控制。的 这项研究的基本原理是ITAF-IRES相互作用是每个EV 71蛋白质合成的核心， 它的复制。因此，这些新知识也将适用于类似的电动汽车。强劲的初步数据 导致三个具体目标：（1）确定有助于病毒复制的IRES元件的结构;（2） 揭示协调病毒翻译的拮抗ITAF的详细分子相互作用;和（3）鉴定 病毒小RNA抑制翻译的机制。对于目标1，遗传学，化学探测，核磁共振， SAXS和细胞测定将确定有助于EV 71翻译的IRES结构元件。对于目标2， 结构、生物物理和病毒学分析将揭示蛋白质和RNA的构象变化，以及功能变化。 在hnRNP A1和AUF 1与IRES SLII区域结合后， 控制对于目标3，结构、生物化学和病毒学分析将揭示结构和功能效应。 vsRNA 1对拮抗性hnRNP A1-和AUF 1-RNP的影响。该建议是创新的，采用了一种在- 结合结构生物化学，生物物理学和病毒学研究的综合计划，承诺提供 这些重大突破有助于更好地理解导致EV 71发病机制的病毒-宿主相互作用。的 这项研究意义重大，因为它有望为RNP和RNA-RNA相互作用提供前所未有的见解， 导致EV发病的因素。这将反过来为鉴定新的抗病毒药物提供基础 针对EV 71和EV 68，其最近被NIH指定为新兴病原体。