Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
J Virol. 2023 Mar 30;97(3):e0186522. doi: 10.1128/jvi.01865-22. Epub 2023 Feb 27.
Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable β5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的复制强烈影响细胞代谢，并迅速导致细胞病变效应（CPE）。病毒诱导修饰的特征是抑制细胞 mRNA 的翻译，并将细胞翻译机制重新定向到病毒特异性蛋白的合成。SARS-CoV-2 的多功能非结构蛋白 1（nsp1）是主要的毒力因子，也是导致翻译关闭的关键因素。在这项研究中，我们应用了广泛的病毒学和结构方法来进一步分析 nsp1 的功能。单独表达这种蛋白就足以引起 CPE。然而，我们选择了几个表现出非致病变表型的 nsp1 突变体。在三个簇中检测到了减弱突变，这些簇位于 C 末端螺旋、结构域的一个环中和 nsp1 的无序和结构片段的连接处。基于 NMR 的野生型 nsp1 及其突变体的分析并未证实 X 射线结构所提出的稳定β5 链的存在。在溶液中，这种蛋白质似乎以动态构象存在，这是其在 CPE 发展和病毒复制中发挥功能所必需的。NMR 数据还表明 N 端和 C 端结构域之间存在动态相互作用。鉴定的 nsp1 突变使该蛋白不具有细胞毒性且不能诱导翻译关闭，但不会对病毒的细胞病变毒性产生有害影响。SARS-CoV-2 的 nsp1 是一种多功能蛋白，可修饰细胞内环境以满足病毒复制的需要。它负责翻译关闭的发展，其单独表达足以引起细胞病变效应（CPE）。在这项研究中，我们选择了广泛的表现出非致病变表型的 nsp1 突变体。通过病毒学和结构方法对聚集在 nsp1 的三个不同片段中的减弱突变进行了广泛的表征。我们的数据强烈表明 nsp1 结构域之间存在相互作用，这是该蛋白在 CPE 发展中发挥功能所必需的。大多数突变使 nsp1 不具有细胞毒性且不能诱导翻译关闭。它们大多数不影响病毒的生存能力，但它们确实降低了在诱导和信号转导 I 型干扰素的细胞中复制的速度。这些突变及其组合，特别是可以用于开发具有减弱表型的 SARS-CoV-2 变体。
