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Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication

Abstract : 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 presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.
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https://hal-amu.archives-ouvertes.fr/hal-03411659
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Submitted on : Tuesday, November 2, 2021 - 2:23:14 PM
Last modification on : Friday, April 1, 2022 - 3:54:42 AM
Long-term archiving on: : Thursday, February 3, 2022 - 7:07:00 PM

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Natacha Ogando, Priscila El Kazzi, Jessika Zevenhoven-Dobbe, Brenda Bontes, Alice Decombe, et al.. Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2021, ⟨10.1101/2021.05.17.444407⟩. ⟨hal-03411659⟩

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