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-5' exoribonuclease (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. Significance StatementThe ongoing SARS-CoV-2 pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure-function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different {beta}-CoVs, we characterized the key conserved residues of nsp14 (N7-guanine)-methyltransferase, a poorly defined subunit of the CoV mRNA-synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in {beta}-CoV replication, while identifying two residues that are critical for the replication of all four {beta}-CoVs tested, including SARS-CoV-2.