Structural and functional insights into the 20-O-methyltransferase of SARS-CoV-2  

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

机构地区：[1]State Key Laboratory of Virology,RNA Institute,College of Life Sciences and Frontier Science Center for Immunology and Metabolism,Wuhan University,Wuhan,430072,China [2]Animal Bio-Safety Level Ⅲ Laboratory/Institute for Vaccine Research,Wuhan University School of Medicine,Wuhan,430071,China

出　　处：《Virologica Sinica》2024年第4期619-631,共13页中国病毒学（英文版）

基　　金：supported by grants from the National Natural Science Foundation of China(82372223 and 82172243);the National Key R&D Program of China(2021YFF0702004);the Fundamental Research Funds for the Central Universities of China(2042022dx0003).

摘　　要：A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16(nsp16),20-Omethyltransferase(20-O-MTase),to cap their RNAs through ribose 20-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 20-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 Sadenosyl-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 20-O-MTase activity.Additionally,we utilized an in vitro biochemical platform to screen potential inhibitors targeting 20-O-MTase activity.Overall,our results significantly enhance the understanding of viral 20-O methylation process and mechanism,providing valuable targets for antiviral drug development.

关 键 词：SARS-CoV-2 nsp16/nsp10 20-O-Methyltransferase 3-Dimensional structure Molecular mechanism INHIBITORS 

分 类 号：R563.1[医药卫生—呼吸系统]