Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是一种 β-CoV，最近作为人类病原体出现，是 COVID-19 大流行的病原体。该病毒如何操纵宿主细胞机制以促进感染的分子框架仍不清楚。在这里，我们重点关注 SARS-CoV-2 NSP1，它被认为是一种毒力因子，通过直接结合人类核糖体来抑制蛋白质合成。我们通过生化方法证明 NSP1 抑制模型人类和 SARS-CoV-2 信使 RNA (mRNA) 的翻译。 NSP1 特异性结合小核糖体亚基 (40S)，这是翻译抑制所必需的。使用单分子荧光测定实时监测 NSP1-40S 亚基结合，我们确定真核翻译起始因子 (eIF) 在没有 mRNA 的情况下变构调节 NSP1 与核糖体前起始复合物的相互作用。我们进一步阐明，NSP1 与起始密码子下游的 RNA 片段竞争结合 40S 亚基，并且该蛋白质无法与 mRNA 上组装的 80S 核糖体快速结合。总的来说，我们的研究结果支持这样一个模型：至少两个 β-CoV 亚属中的病毒 NSP1 蛋白与 40S 亚基的开放头构象相关，通过阻止 mRNA 在进入通道内的调节来抑制翻译的早期步骤。