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

• Published in: Virologica Sinica 2024-08, Vol.39 (4), p.619-631
• Main Authors: Deng, Jikai, Gong, Feiyu, Li, Yingjian, Tan, Xue, Liu, Xuemei, Yang, Shimin, Chen, Xianying, Wang, Hongyun, Liu, Qianyun, Shen, Chao, Zhou, Li, Chen, Yu
• Format: Article
• Language: English
• Subjects: 2′-O-Methyltransferase; 3-Dimensional structure; Betacoronavirus - enzymology; Betacoronavirus - genetics; COVID-19 - virology; Humans; Inhibitors; Methylation; Methyltransferases - chemistry; Methyltransferases - genetics; Methyltransferases - metabolism; Middle East Respiratory Syndrome Coronavirus - enzymology; Middle East Respiratory Syndrome Coronavirus - genetics; Models, Molecular; Molecular mechanism; nsp16/nsp10; Protein Binding; RNA, Viral - genetics; RNA, Viral - metabolism; S-Adenosylmethionine - metabolism; SARS-CoV-2; SARS-CoV-2 - enzymology; SARS-CoV-2 - genetics; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism; Viral Regulatory and Accessory Proteins
• ISSN: 1995-820X; 1674-0769; 1995-820X
• DOI: 10.1016/j.virs.2024.07.001

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 nsp10 K58T resulted in a restriction of functional activation of MERS-CoV nsp16.•The cofactor nsp10 enhances the binding of nsp16 to the substrate RNA and the methyl donor SAM.•The cofactor nsp10 interacts with specific residues of nsp16 to effectively regulate and stabilize the binding pockets.•Sinefungin, 7MeGpppA, and SAH, exhibit significant inhibitory effects on SARS-CoV-2 2′-O-methyltransferase activity.