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

Autor:	Ogando NS; Department of Medical Microbiology, Leiden University Medical Center 2333 ZA Leiden, The Netherlands., El Kazzi P; Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Aix-Marseille Université 13288 Marseille, France., Zevenhoven-Dobbe JC; Department of Medical Microbiology, Leiden University Medical Center 2333 ZA Leiden, The Netherlands., Bontes BW; Department of Medical Microbiology, Leiden University Medical Center 2333 ZA Leiden, The Netherlands., Decombe A; Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Aix-Marseille Université 13288 Marseille, France., Posthuma CC; Department of Medical Microbiology, Leiden University Medical Center 2333 ZA Leiden, The Netherlands., Thiel V; Institute of Virology and Immunology (IVI) 3350 Bern, Switzerland.; De partment of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern 3012 Bern, Switzerland., Canard B; Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Aix-Marseille Université 13288 Marseille, France.; European Virus Bioinformatics Center (EVBC), Jena 07743, Germany., Ferron F; Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Aix-Marseille Université 13288 Marseille, France.; European Virus Bioinformatics Center (EVBC), Jena 07743, Germany., Decroly E; Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Aix-Marseille Université 13288 Marseille, France; E.J.Snijder@lumc.nl Etienne.decroly@univ-amu.fr., Snijder EJ; Department of Medical Microbiology, Leiden University Medical Center 2333 ZA Leiden, The Netherlands; E.J.Snijder@lumc.nl Etienne.decroly@univ-amu.fr.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Dec 07; Vol. 118 (49).
DOI:	10.1073/pnas.2108709118
Abstrakt:	As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' 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 nsp14's 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. Competing Interests: The authors declare no competing interest. (Copyright © 2021 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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