Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in betacoronavirus replication
As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5 exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3-to-5exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is assumed to be involved in viral mRNA capping. Here, we first revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between different betacoronaviruses (beta-CoVs). In this study, we identified several residues likely to be involved in the formation of the catalytic pocket of N7MTase, which presents a fold that is distinct from the Rossmann fold observed in most known MTases. Next, for multiple beta-CoVs, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity and viral replication in cell culture. For SARS-CoV and Middle East respiratory syndrome-CoV, most of the engineered mutations abolished the N7-MTase function, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into beta-CoV genomes, we identified two substitutions (R310A and F426A in SARS-CoV) that abrogated viral progeny production and one mutation (H424A) that yielded a crippled phenotype across all beta-CoVs tested. Our results identify the N7-MTase as a critical enzyme for beta-CoV replication and defined key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.