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

• 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
• 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' 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

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.

病毒依赖于宿主细胞的翻译机构，因此，感染的结果取决于宿主通过先天免疫反应抑制病毒翻译的能力与病毒抵消它们并篡夺翻译机构的能力之间的平衡。冠状病毒病2019(新冠肺炎)是由严重急性呼吸综合征冠状病毒2(SARS-CoV2)引起的，SARS-CoV2是冠状病毒科的一种贝塔冠状病毒，还包括临床上重要的SARS-CoV和MERS-CoV。在感染过程中，冠状病毒(CoV)利用双重策略抑制细胞mRNAs的翻译和诱导其降解，同时选择性地使病毒mRNAs能够进入细胞翻译装置。这一策略是由病毒非结构蛋白Nsp1介导的，Nsp1与40S核糖体亚基结合，通过两种机制诱导宿主蛋白质合成的停止：通过直接停止细胞mRNAs的翻译，以及通过诱导它们的内切和随后的降解。冠状病毒基因组和所有亚基因组的5‘-非翻译区都含有一个共同的~60-70个核苷酸长的元件，其中包括茎环SL1，它使病毒mRNAs对Nsp1介导的翻译抑制和内切具有抵抗力。这些过程对病毒复制和致病是至关重要的，尽管它们已成为潜在的靶点，可用于广泛的抗冠状病毒应用，但它们仍然知之甚少：启动基因组和亚基因组冠状病毒mRNAs的因子要求和分子细节从未确定，病毒逃避Nsp1介导的翻译关闭的机制尚不清楚，负责Nsp1诱导的细胞mRNAs裂解的内切酶及其募集到核糖体复合体的机制尚不清楚。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并使用一系列生化技术解剖它们的各个阶段来阐明这些过程的机制。在目标1中，我们将通过确定完整的必需因子集，表征它们在这一过程中的作用机制，并通过确定这些mRNAs负责冠状病毒启动过程的独特方面的性质，来全面概述对基因组和亚基因组SARV-CoV2 mRNAs的启动。在目标2中，我们建议描述Nsp1对细胞mRNAs启动的各个阶段的影响，并研究Nsp1介导的翻译关闭的病毒逃避机制。目的3将着重于鉴定介导Nsp1诱导宿主细胞mRNAs切割的细胞内切酶，鉴定其作用机制，并鉴定病毒mRNAs中抵抗内切核裂解的元件。