All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein.
All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein.
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DOI：
10.1128/jvi.01865-22
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发表时间：
2023-03-30
影响因子：
5.4
通讯作者：
Frolova, Elena I.
中科院分区：
文献类型：
--
作者：
Frolov, Ilya;Agback, Tatiana;Palchevska, Oksana;Dominguez, Francisco;Lomzov, Alexander;Agback, Peter;Frolova, Elena I.
关键词：
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，导致细胞病变效应 (CPE) 快速发展。病毒诱导修饰的特点是抑制细胞 mRNA 的翻译以及将细胞翻译机制重定向至病毒特异性蛋白质的合成。 SARS-CoV-2 的多功能非结构蛋白 1 (nsp1) 是主要毒力因子，也是翻译关闭发展的关键贡献者。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 功能。研究发现，仅该蛋白的表达就足以引起 CPE。然而，我们选择了几个表现出非细胞病变表型的 nsp1 突变体。在三个簇中检测到减毒突变，分别位于 C 末端螺旋、结构化域的一个环以及 nsp1 无序和结构化片段的连接处。对野生型 nsp1 及其突变体的基于 NMR 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，该蛋白质似乎以动态构象存在，这是其在 CPE 发育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。已鉴定的 nsp1 突变使该蛋白无细胞毒性且无法诱导翻译关闭，但它们不会对病毒细胞致病性产生有害影响。重要性 SARS-CoV-2 的 nsp1 是一种多功能蛋白，可以改变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，并且其单独表达足以引起细胞病变效应（CPE）。在这项研究中，我们选择了多种表现出非细胞病变表型的 nsp1 突变体。减毒突变聚集在 nsp1 的三个不同片段中，通过病毒学和结构方法进行了广泛的表征。我们的数据强烈表明 nsp1 结构域之间存在相互作用，这是 CPE 发育中蛋白质功能所必需的。大多数突变使 nsp1 无细胞毒性并且无法诱导翻译关闭。它们中的大多数并不影响病毒的活力，但它们确实降低了具有 I 型 IFN 诱导和信号转导能力的细胞的复制速率。这些突变及其组合尤其可用于开发具有减毒表型的 SARS-CoV-2 变体。
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. IMPORTANCE 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.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
3.7
作者：
Agback T;Dominguez F;Frolov I;Frolova EI;Agback P
通讯作者：
Agback P
影响因子：
6.7
作者：
Huang C;Lokugamage KG;Rozovics JM;Narayanan K;Semler BL;Makino S
通讯作者：
Makino S
影响因子：
28.3
作者：
Gorbalenya, Alexander E.;Baker, Susan C.;Ziebuhr, John
通讯作者：
Ziebuhr, John
DOI：
10.1046/j.1432-1327.1998.2560001.x
发表时间：
1998-08-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Markley, JL;Bax, A;Wüthrich, K
通讯作者：
Wüthrich, K