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

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.