Mechanisms of SARS-CoV2 translation initiation and shut-off of cellular protein synthesis

SARS-CoV2翻译启动和细胞蛋白质合成关闭的机制

基本信息

批准号：
10609872
负责人：
CHRISTOPHER Ulrich Tristram HELLEN
金额：
$ 24.36万
依托单位：
SUNY DOWNSTATE MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-14 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10609872
关键词：
2019-nCoV 5&apos Untranslated Regions Address Agreement Animals Binding Biochemical Biological Assay C-terminal COVID-19 Cells Clinical Complement Complex Coronavirus Coupled Disease Elements Endoribonucleases Enzymes Equilibrium Event Family Foundations Future Genomic Segment Genomics Human In Vitro Individual Infection Innate Immune Response Investigation Knowledge Length Mediating Messenger RNA Middle East Respiratory Syndrome Coronavirus Molecular Mutation Analysis Nucleotides Outcome Peptide Initiation Factors Pharmaceutical Preparations Population Positioning Attribute Process Property Protein Biosynthesis Proteins RNA Reporting Repression Resistance Ribosomes Role SARS coronavirus Source Structure Techniques Testing Therapeutic Trans-Activators Translation Initiation Translation Process Translational Repression Translations Viral Viral Nonstructural Proteins Viral Pathogenesis Virus Virus Replication betacoronavirus drug development endonuclease experimental study in vivo inhibitor therapy insight mRNA Transcript Degradation reconstitution recruit stem viral resistance

项目摘要

Viruses depend on the host cell’s translation apparatus and consequently, the outcome of infection is determined by the balance between a host’s ability to repress viral translation via innate immune responses, and viruses’ abilities to counteract them and usurp the translation apparatus. Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), a beta coronavirus of the family Coronaviridae that also includes the clinically important SARS-CoV and MERS- CoV. During infection, coronaviruses (CoVs) utilize a dual strategy of suppressing translation and inducing degradation of cellular mRNAs while selectively enabling viral mRNAs to gain access to the cellular translation apparatus. This strategy is mediated by the viral non-structural protein Nsp1 that binds to 40S ribosomal subunits and induces a shutdown of host protein synthesis by two mechanisms: by direct stalling of translation of cellular mRNAs, and by inducing their endonucleolytic cleavage and subsequent degradation. 5’- untranslated regions of CoV genomic and all subgenomic mRNAs contain a common ~60-70 nucleotide-long element that includes the stem-loop SL1 that confers resistance of viral mRNAs to Nsp1-mediated translational suppression and endonucleolytic cleavage. These processes are critical for viral replication and pathogenesis and although they have emerged as potential targets for chemotherapeutic inhibitors that could have broad anti-coronaviral application, they remain poorly understood: the factor requirements and molecular details of initiation on genomic and subgenomic CoV mRNAs have never been determined, the mechanism of viral evasion of Nsp1-mediated translational shut-off is obscure, and the endonuclease that is responsible for Nsp1- induced cleavage of cellular mRNAs as well as the mechanism of its recruitment to ribosomal complexes are unknown. We propose to elucidate the mechanisms of these processes by recapitulating them in vitro using individual purified translational components and dissecting their individual stages using an array of biochemical techniques. In Aim 1, we will obtain a comprehensive overview of initiation on genomic and subgenomic SARV-CoV2 mRNAs by determining the complete set of required factors, characterizing the mechanisms by which they act in this process, and by identifying properties of these mRNAs that are responsible for unique aspects of the CoV initiation process. In Aim 2, we propose to characterize the influence of Nsp1 on all stages of initiation on cellular mRNAs and to investigate the mechanism of viral evasion of Nsp1-mediated translational shut-off. Aim 3 will focus on identification of the cellular endonuclease that mediates Nsp1- induced cleavage of host cell's mRNAs, characterization of the mechanism of its action, and identification of elements in viral mRNAs that confer resistance to endonucleolytic cleavage.

病毒取决于宿主细胞的翻译设备，因此，感染的结果取决于宿主通过先天免疫反应反映病毒翻译的能力之间的平衡，以及病毒抵消它们并篡夺翻译设备的能力。 2019年冠状病毒病（COVID-19）是由严重的急性呼吸综合征冠状病毒2（SARS-COV2）引起的，这是家族冠状病毒的β冠状病毒，其中还包括临床上重要的SARS-COV和MERS-COV。在感染期间，冠状病毒（COVS）采用了抑制翻译和诱导细胞mRNA降解的双重策略，同时有选择地使病毒mRNA能够进入细胞翻译设备。该策略是由与40S核糖体亚基结合的病毒非结构蛋白NSP1介导的，并通过两种机制诱导宿主蛋白质合成的关闭：通过直接存储细胞mRNA的翻译，并诱导其内核分解性裂解和随后的脱位。 COV基因组和所有亚基因组mRNA的5'-未翻译区域都包含一个〜60-70个核刺元的元素，其中包括茎环SL1，该元素赋予病毒mRNA对NSP1介导的翻译抑制和内核分解裂解的耐药性。 These processes are critical for viral replication and pathogenesis and although they have emerged as potential targets for chemotherapeutic inhibitors that could have broad anti-coronaviral application, they remain poorly understood: the factor requirements and molecular details of initiation on genomic and subgenomic CoV mRNAs have never been determined, the mechanism of viral evolution of Nsp1-mediated translational shut-off is obscure, and the endonuclease that负责NSP1诱导的细胞mRNA裂解以及其募集到核糖体复合物的机制尚不清楚。我们建议通过使用单个纯化的翻译成分在体外概括这些过程的机制，并使用一系列的生化技术来解剖其各个阶段。在AIM 1中，我们将通过确定所需因素的完整集合来获得有关基因组和亚基因组SARV-COV2 mRNA启动的全面概述，以表征它们在此过程中起作用的机制，并通过识别这些mRNA的性质，这些特性负责COV启动过程的独特方面。在AIM 2中，我们建议表征NSP1对启动的所有阶段对细胞mRNA的影响，并研究NSP1介导的翻译封闭的病毒演化机制。 AIM 3将集中于鉴定介导NSP1诱导的宿主细胞mRNA裂解，其作用机理的表征以及病毒mRNA中元素的鉴定，以介导了宿主细胞的mRNA，并鉴定了对内核水解裂解的抗性。