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反应抑制病毒翻译的能力和病毒对抗它们并篡夺翻译装置的能力之间的平衡。冠状病毒病2019 （COVID-19）由严重急性呼吸综合征冠状病毒2 （SARS-CoV2）引起，SARS-CoV2是冠状病毒科的一种β冠状病毒，冠状病毒科还包括临床重要的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，它与40S核糖体亚基结合，并通过两种机制诱导宿主蛋白合成的关闭：直接停止细胞mrna的翻译，以及诱导其核内溶裂解和随后的降解。冠状病毒基因组和所有亚基因组mrna的5 ' -非翻译区含有一个共同的~60-70核苷酸长的元件，其中包括茎环SL1，该元件赋予病毒mrna对nsp1介导的翻译抑制和核内溶分裂的抗性。这些过程对病毒复制和发病机制至关重要，尽管它们已成为化疗抑制剂的潜在靶点，可能具有广泛的抗冠状病毒应用，但对它们的了解仍然很少：基因组和亚基因组冠状病毒mrna起始的因子需求和分子细节尚未确定，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mrna切割的内切酶及其向核糖体复合体募集的机制尚不清楚。我们建议阐明这些过程的机制，通过在体外使用单个纯化的翻译成分概括它们，并使用一系列生化技术剖析它们的各个阶段。在目标1中，我们将通过确定全套所需因子，描述它们在这一过程中起作用的机制，并通过鉴定这些mrna的特性来对冠状病毒起始过程的独特方面负责，从而全面概述基因组和亚基因组SARV-CoV2 mrna的起始。在Aim 2中，我们提出表征Nsp1对细胞mrna起始的所有阶段的影响，并研究病毒逃避Nsp1介导的翻译关闭的机制。目的3将着重于鉴定介导Nsp1诱导的宿主细胞mrna裂解的细胞内切酶，表征其作用机制，以及鉴定病毒mrna中赋予抗内核裂解能力的元素。

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.