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：
10354475
负责人：
CHRISTOPHER Ulrich Tristram HELLEN
金额：
$ 20.23万
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 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10354475
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 Genomics Human In Vitro Individual Infection Innate Immune Response Investigation Knowledge Length Light Mediating Messenger RNA Middle East Respiratory Syndrome Coronavirus Molecular Mutation Analysis Nonstructural Protein 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 Trans-Activators Translation Initiation Translation Process Translations Viral Viral Pathogenesis Virus Virus Replication base betacoronavirus drug development endonuclease experimental study in vivo inhibitor 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.

病毒依赖于宿主细胞的翻译装置，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抗病毒翻译并篡夺翻译装置的能力之间的平衡，2019 年冠状病毒病。 COVID-19）是由严重急性呼吸综合征冠状病毒 2（SARS-CoV2）引起的，它是冠状病毒家族的一种 β 冠状病毒，该家族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) 利用抑制翻译和诱导细胞 mRNA 降解的双重策略，同时选择性地使病毒 mRNA 能够进入细胞翻译装置，该策略由与 40S 核糖体结合的病毒非结构蛋白 Nsp1 介导。亚基并通过两种机制诱导宿主蛋白质合成的关闭：直接停止细胞 mRNA 的翻译，以及诱导其核酸内切裂解和随后的 5'- 降解。冠状病毒基因组和所有亚基因组 mRNA 的非翻译区都含有一个共同的约 60-70 个核苷酸长的元件，其中包括赋予病毒 mRNA 对 Nsp1 介导的翻译抑制和核酸内切裂解的抗性的茎环 SL1，这些过程对于病毒复制至关重要。和发病机制，尽管它们已成为具有广泛抗冠状病毒应用的化疗抑制剂的潜在靶点，但它们仍然知之甚少：其因素要求和分子细节基因组和亚基因组 CoV mRNA 的启动尚未确定，Nsp1 介导的翻译关闭的病毒逃避机制尚不清楚，负责 Nsp1 诱导的细胞 mRNA 裂解及其招募机制的核酸内切酶我们建议通过使用单独的纯化翻译组件在体外重现这些过程并使用一系列生化技术剖析它们的各个阶段来阐明这些过程的机制。在目标 1 中，我们将通过确定所需因素的完整集合、表征它们在此过程中的作用机制以及识别这些负责的 mRNA 的特性，获得基因组和亚基因组 SARV-CoV2 mRNA 启动的全面概述。在 Aim 2 中，我们建议表征 Nsp1 对细胞 mRNA 启动的所有阶段的影响，并研究 Nsp1 介导的翻译关闭的病毒逃避机制。第 3 部分将重点关注介导 Nsp1 诱导的宿主细胞 mRNA 裂解的细胞核酸内切酶的鉴定、其作用机制的表征以及病毒 mRNA 中赋予核酸内切裂解抗性的元件的鉴定。