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

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应答抑制病毒翻译的能力与病毒抵消它们并篡夺翻译装置的能力之间的平衡。2019冠状病毒病（COVID-19）是由严重急性呼吸道综合征冠状病毒2（SARS-CoV 2）引起的，这是冠状病毒科的一种β冠状病毒，还包括临床上重要的SARS-CoV和MERS-CoV。在感染期间，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。这种策略是由病毒非结构蛋白Nsp 1介导的，Nsp 1与40 S核糖体亚基结合，并通过两种机制诱导宿主蛋白质合成的关闭：通过直接停止细胞mRNA的翻译，以及通过诱导其内切核酸裂解和随后的降解。CoV基因组和所有亚基因组mRNAs的5 '-非翻译区含有一个共同的约60-70个核苷酸长的元件，包括茎环SL 1，其赋予病毒mRNAs对Nsp 1介导的翻译抑制和核酸内切酶切割的抗性。这些过程对于病毒复制和发病机制至关重要，尽管它们已成为可能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：基因组和亚基因组CoV mRNA起始的因子要求和分子细节从未被确定，病毒逃避Nsp 1介导的翻译关闭的机制尚不清楚，而负责Nsp 1诱导的细胞mRNA切割的核酸内切酶以及其募集到核糖体复合物的机制尚不清楚。我们建议通过在体外使用单个纯化的翻译组分和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，我们将获得一个全面的概述启动基因组和亚基因组SARV-CoV 2的mRNA，通过确定所需的因素，表征他们在这一过程中发挥作用的机制，并通过确定这些mRNA的特性，负责独特的方面的冠状病毒启动过程。在目标2中，我们提出表征Nsp 1对细胞mRNA起始的所有阶段的影响，并研究病毒逃避Nsp 1介导的翻译关闭的机制。目标3将集中于鉴定介导Nsp 1诱导的宿主细胞mRNA裂解的细胞核酸内切酶，表征其作用机制，并鉴定赋予对核酸内切裂解抗性的病毒mRNA中的元件。

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.