RNA viruses have several mechanisms to modify the viral RNA genome to protect from host immune surveillance. SARS-CoV-2, the etiological agent of COVID-19, and other coronaviruses encode capping proteins in its genome that modify the 5’ untranslated region of the viral genome and mRNA. Capping viral RNAs promotes translation, prevents degradation, and reduces the activation of host immune responses. This process is initiated by the viral proteins nsp13 and nsp12, which cleave the 5’ phosphate group and transfer a GMP from GTP to the 5 end of the nascent (+)ssRNA. The nsp14-nsp10 complex then catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the N7 position of the GMP cap, generating Cap-0. Finally, the nsp16-nsp10 complex utilizes SAM to catalyze the 2-O-methylation of the first ribonucleotide, generating Cap-1. Several structures of the nsp16-nsp10 complex have been solved previously for SARS-CoV, MERS and SARS-CoV-2 with SAM, Cap-0 and Cap-0-RNA bound to the complex as well as the products of the reactions. Recently, we determined the first structure of nsp16-nsp10 with Cap-0-RNA and Cap-1 RNA, revealing that Mn2+ coordinates the first four nucleotides of the bound RNA to orient the 2’-OH of the ribose of the first nucleotide toward the methyl group of SAM for catalysis. Herein, we also show the first apo-crystal structure of nsp16 determined in which neither SAM nor RNA substrates bound. This new structure and comparison to structures with substraces and products bound, will reveal critical movements of the enzyme for binding substates. This study shows an important set of structures of the viral 2’-O-methyltransferases revealing unique features of this complex that could be used for molecular dynamics studies and designing coronavirus-specific inhibitors.
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no.DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). NIAID/NIH/HHS contract HHSN272201700060C, Chicago Biomedical Consortium COVID Response Award CR-003, and NCI CCSG P30CA060553.
