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: 10392690
• 负责人: Gary A. Brewer
• 金额: $ 0.89万
• 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/10392690
• 关键词: 5' Untranslated Regions; Antiviral Agents; Binding; Biochemical; Biological Assay; Biological Process; Biophysics; Cells; Cellular Assay; Chemicals; Complex; Data; Elements; Enterovirus; Enterovirus 71; Foundations; Gene Expression; Genes; Genetic; Genome; Goals; Infection; Integration Host Factors; Internal Ribosome Entry Site; Knowledge; Lead; Mission; Modeling; Molecular; Molecular Conformation; National Institute of General Medical Sciences; Pathogenesis; Pathogenicity; Phylogenetic Analysis; Protein Biosynthesis; Protein Conformation; Proteins; Public Health; RNA; RNA Conformation; RNA-Binding Proteins; Research; Ribonucleoproteins; Ribosomes; Small RNA; Structural Biochemistry; Structure; Translations; United States National Institutes of Health; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Replication; advanced disease; emerging pathogen; hnRNP A1; innovation; insight; novel; programs; recruit; stem; 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) 通过其协调蛋白质合成和基因组复制 5'UTR，预计折叠成六个茎环。茎环 (SL) I 促进病毒基因组复制； SLII- VI 充当独立于 cap 的翻译的基本 IRES 单元。几种宿主RNA结合蛋白（称为 作为ITAF），被招募到IRES以进行有效的病毒蛋白合成。当前的教条支持一个模型 其中ITAF以控制核糖体进入的构象稳定或不稳定IRES结构。这 基本知识差距是对 ITAF 与 ITAF 进行功能互动的机制的理解。 5'UTR 组织 IRES 结构。这些知识对于揭示电动汽车的潜在机制至关重要 重定向宿主因素以维持感染。长期目标是更好地了解分子机制 EV 颠覆宿主因子以调节病毒基因表达和复制的机制。总体目标—— 该提案的目的是阐明 ITAF 如何与病毒 IRES 功能相互作用以控制翻译。 EV71 和 ITAF hnRNP A1 和 AUF1 是优秀的模型，因为它们结合相同的 IRES 结构域 SLII 反向微调 EV71 翻译水平。此外，vsRNA1，一种源自病毒的小RNA，产生 通过 Dicer 切割 SLII IRES 区域，进一步抑制 EV71 翻译。我们的中心假设是 保守的病毒RNA元件通过组装独特且拮抗的核糖体来微调EV71翻译水平 核蛋白 (RNP) 复合物和 vsRNA1 竞争这些相互作用，以实现更严格的控制。这 这项研究的基本原理是 ITAF-IRES 相互作用是每个 EV71 蛋白合成的核心，并且 它的复制。因此，这一新知识也将被证明适用于类似的电动汽车。强有力的初步数据 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性 ITAF 的详细分子相互作用； (3) 识别 病毒小RNA抑制翻译的机制。对于 Aim1、系统发育学、化学探测、NMR、 SAXS 和细胞检测将定义有助于 EV71 翻译的 IRES 结构元件。对于目标 2， 结构、生物物理和病毒学检测将揭示蛋白质和 RNA 构象变化以及功能 hnRNP A1 和 AUF1 与 IRES SLII 区域结合后发生的变化，这对于翻译至关重要 控制。对于目标 3，结构、生化和病毒学测定将揭示结构和功能效应 vsRNA1 对拮抗 hnRNP A1- 和 AUF1-RNP 的影响。该提案通过采用内部技术而具有创新性 结合结构生物化学、生物物理学和病毒学研究的综合计划，有望提供 更好地理解导致 EV71 发病机制的病毒与宿主相互作用的重大突破。这 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。 有助于 EV 发病机制的因素。这反过来将为鉴定新的抗病毒药物提供基础 针对 EV71 和 EV68，NIH 最近将其指定为新出现的病原体。

项目成果

The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

• DOI: 10.1016/j.jbc.2023.103028
• 发表时间: 2023-04
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Mackeown, Matthew;Kung, Yu-An;Davila-Calderon, Jesse;Ford, William P.;Luo, Le;Henry, Barrington;Li, Mei-Ling;Brewer, Gary;Shih, Shin-Ru;Tolbert, Blanton S.
• 通讯作者: Tolbert, Blanton S.

IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex.

• DOI: 10.1038/s41467-020-18594-3
• 发表时间: 2020-09-22
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Davila-Calderon J;Patwardhan NN;Chiu LY;Sugarman A;Cai Z;Penutmutchu SR;Li ML;Brewer G;Hargrove AE;Tolbert BS
• 通讯作者: Tolbert BS

Idiosyncrasies of hnRNP A1-RNA recognition: Can binding mode influence function.

• DOI: 10.1016/j.semcdb.2018.04.001
• 发表时间: 2019-03
• 期刊: Seminars in cell & developmental biology
• 影响因子: 7.3
• 作者: Levengood JD;Tolbert BS
• 通讯作者: Tolbert BS

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

• DOI: 10.1128/mbio.02717-21
• 发表时间: 2022-02-22
• 期刊: mBio
• 影响因子: 6.4
• 作者: Kung YA;Chiang HJ;Li ML;Gong YN;Chiu HP;Hung CT;Huang PN;Huang SY;Wang PY;Hsu TA;Brewer G;Shih SR
• 通讯作者: Shih SR

Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

• DOI: 10.1002/prot.26058
• 发表时间: 2021-07
• 期刊: Proteins
• 影响因子: 2.9