Biochemical characterization of naturally occurring mutations in SARS-CoV-2 RNA-dependent RNA polymerase.
RNA‐dependent RNA polymerase (RdRp)
SARS‐CoV‐2
mutations
phenotypic effect
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
Sep 2024
Sep 2024
Historique:
revised:
06
06
2024
received:
24
02
2024
accepted:
21
06
2024
medline:
15
8
2024
pubmed:
15
8
2024
entrez:
15
8
2024
Statut:
ppublish
Résumé
Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.
Substances chimiques
Coronavirus RNA-Dependent RNA Polymerase
EC 2.7.7.48
NSP12 protein, SARS-CoV-2
EC 2.7.7.48
NS8 protein, SARS-CoV-2
0
NSP7 protein, SARS-CoV-2
EC 2.7.7.48
Viral Nonstructural Proteins
0
RNA-Dependent RNA Polymerase
EC 2.7.7.48
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e5103Subventions
Organisme : National Institute of Virology and Bacteriology
ID : LX22NPO5103
Organisme : European Union - Next Generation EU and by Czech Science Foundation
ID : 22-17118S
Informations de copyright
© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
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