Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2

Jikai Deng; Feiyu Gong; Yingjian Li; Xue Tan; Xuemei Liu; Shimin Yang; Xianying Chen; Hongyun Wang; Qianyun Liu; Chao Shen; Li Zhou; Yu Chen

doi:10.1016/j.virs.2024.07.001

August 2024

Jikai Deng, Feiyu Gong, Yingjian Li, Xue Tan, Xuemei Liu, Shimin Yang, Xianying Chen, Hongyun Wang, Qianyun Liu, Chao Shen, Li Zhou and Yu Chen. Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2[J]. Virologica Sinica, 2024, 39(4): 619-631. doi: 10.1016/j.virs.2024.07.001

Citation: Jikai Deng, Feiyu Gong, Yingjian Li, Xue Tan, Xuemei Liu, Shimin Yang, Xianying Chen, Hongyun Wang, Qianyun Liu, Chao Shen, Li Zhou, Yu Chen. Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2 .VIROLOGICA SINICA, 2024, 39(4) : 619-631. http://dx.doi.org/10.1016/j.virs.2024.07.001

SARS-CoV-2 2'-O-甲基转移酶的结构和功能洞察

邓积凯 ^a ,
龚飞宇 ^a ,
李颖健 ^a ,
谭雪 ^a ,
刘雪梅 ^a ,
杨世敏 ^a ,
陈仙莹 ^a ,
王虹运 ^a ,
刘乾运 ^a ,
沈超 ^a ,
周立 ^a,b ,
陈宇 ^a,b,,

cstr: 32224.14.j.virs.2024.07.001

a. 武汉大学生命科学学院，病毒学国家重点实验室;
b. 武汉大学动物实验中心/ABSL-III实验室/疫苗研究院

通讯作者： 陈宇, chenyu@whu.edu.cn
收稿日期： 2023-11-24
录用日期： 2024-07-02

摘要

冠状病毒的一个独特特性是它们利用自身编码的非结构性蛋白16（nsp16），即2'-O-甲基转移酶（2'-O-MTase），通过核糖2'-O-甲基化修饰对其RNA进行加帽反应。这一过程对于维护病毒基因组稳定性、促进有效的翻译以及实现免疫逃逸至关重要。尽管对SARS-CoV-2 nsp16/nsp10的超微结构已有相当进展，但对其分子机制的了解目前还很有限。在本研究中，我们系统性地研究了SARS-CoV-2中nsp16的2'-O-MTase活性，重点关注其对于nsp10刺激的依赖性。我们观察到由于保守的相互作用界面，nsp16与nsp10在不同的冠状病毒之间存在交叉反应性。然而，在SARS-CoV-2 nsp10中的单个残基替换（K58T）限制了MERS-CoV nsp16的功能激活。进一步地，辅因子nsp10有效地增强了nsp16与底物RNA以及甲基供体S-腺苷基-L-甲硫氨酸（SAM）的结合。从机制上讲，nsp10的His-80、Lys-93和Gly-94分别与nsp16的Asp-102、Ser-105和Asp-106相互作用，进而稳定了SAM结合口袋。nsp10的Lys-43与nsp16的Lys-38和Gly-39相互作用，动态调节RNA结合口袋，并促进RNA精确绑定到nsp16/nsp10复合物中。通过评估nsp16/nsp10复合物的构象表位，我们进一步确定了参与2'-O-MTase活性的关键残基。此外，我们利用体外生化平台筛选了针对2'-O-MTase活性的潜在抑制剂。总体来说，我们的结果显著增强了对病毒2'-O甲基化过程和机制的理解，为抗病毒药物开发提供了有价值的靶点。
- SARS-CoV-2
- , 非结构蛋白16/10
- , 2'-O甲基转移酶
- , 三维结构
- , 分子机制
- , 抑制剂

Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2

Jikai Deng ^a ,
Feiyu Gong ^a ,
Yingjian Li ^a ,
Xue Tan ^a ,
Xuemei Liu ^a ,
Shimin Yang ^a ,
Xianying Chen ^a ,
Hongyun Wang ^a ,
Qianyun Liu ^a ,
Chao Shen ^a ,
Li Zhou ^a,b ,
Yu Chen ^a,b,,

a. State Key Laboratory of Virology, RNA Institute, College of Life Sciences and Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, 430072, China;
b. Animal Bio-Safety Level III Laboratory/Institute for Vaccine Research, Wuhan University School of Medicine, Wuhan, 430071, China

Corresponding author: Yu Chen, chenyu@whu.edu.cn
Received Date: 24 November 2023
Accepted Date: 02 July 2024

Abstract

A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2'-O-methyltransferase (2'-O-MTase), to cap their RNAs through ribose 2'-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2'-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2'-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2'-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2'-O methylation process and mechanism, providing valuable targets for antiviral drug development.
- SARS-CoV-2
- , nsp16/nsp10
- , 2'-O-Methyltransferase
- , 3-Dimensional structure
- , Molecular mechanism
- , Inhibitors

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