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Publication . Article . Preprint . 2021

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

Natacha S. Ogando; Priscila El Kazzi; Jessika C. Zevenhoven-Dobbe; Brenda w. Bontes; Alice Decombe; Clara C. Posthuma; Volker Thiel; +4 Authors
Open Access
English
Abstract

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.

Significance The ongoing severe acute respiratory syndrome coronavirus 2 (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 betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)–methyltransferase, a poorly defined subunit of the CoV messenger RNA–synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2.

Subjects by Vocabulary

Medical Subject Headings: viruses virus diseases respiratory tract diseases

Microsoft Academic Graph classification: Rossmann fold Genetics Betacoronavirus biology.organism_classification biology Host cell cytoplasm In silico Mutation medicine.disease_cause medicine Mutagenesis (molecular biology technique) RNA-dependent RNA polymerase Exoribonuclease Middle East respiratory syndrome medicine.disease

Subjects

SARS-CoV, MERS-CoV, SARS-CoV-2, RNA synthesis, mRNA capping, 423, 535, Biological Sciences, Microbiology, 630 Agriculture, 500 Science, 570 Life sciences; biology, 590 Animals (Zoology), 610 Medicine & health, Microbiology SARS-CoV, [SDV]Life Sciences [q-bio], Microbiology SARS-CoV-2, 610 Medicine & health, Multidisciplinary

Funded by
EC| ANTIVIRALS
Project
ANTIVIRALS
European Training Network on Antiviral Drug Development
  • Funder: European Commission (EC)
  • Project Code: 642434
  • Funding stream: H2020 | MSCA-ITN-ETN
Validated by funder
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COVID-19
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