Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in
Amino Acid Sequence
Base Sequence
Binding Sites
Catalytic Domain
Conserved Sequence
Exoribonucleases
/ chemistry
Microbial Viability
Models, Molecular
Nucleotide Motifs
Protein Conformation
RNA, Viral
/ chemistry
RNA-Binding Proteins
Structure-Activity Relationship
Viral Nonstructural Proteins
/ chemistry
Virus Replication
/ genetics
MERS-CoV
RNA synthesis
SARS-CoV
SARS-CoV-2
mRNA capping
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Titre abrégé: Proc Natl Acad Sci U S A
Pays: United States
ID NLM: 7505876
Informations de publication
Date de publication:
07 12 2021
07 12 2021
Historique:
accepted:
16
10
2021
entrez:
30
11
2021
pubmed:
1
12
2021
medline:
15
12
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34845015
pii: 2108709118
doi: 10.1073/pnas.2108709118
pmc: PMC8670481
pii:
doi:
Substances chimiques
RNA, Viral
0
RNA-Binding Proteins
0
Viral Nonstructural Proteins
0
nsp14 protein, SARS coronavirus
EC 2.1.1.56
Exoribonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
Copyright © 2021 the Author(s). Published by PNAS.
Déclaration de conflit d'intérêts
The authors declare no competing interest.
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