A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability.
Antiviral Agents
/ chemistry
Binding Sites
Crystallography, X-Ray
Enterovirus A, Human
/ chemistry
Genome, Viral
Humans
Models, Molecular
Multiprotein Complexes
/ chemistry
Nucleotides
/ chemistry
Protein Conformation
RNA, Viral
/ chemistry
RNA-Dependent RNA Polymerase
/ chemistry
Virus Replication
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
20 02 2020
20 02 2020
Historique:
accepted:
05
12
2019
revised:
30
11
2019
received:
09
09
2019
pubmed:
22
12
2019
medline:
20
3
2020
entrez:
22
12
2019
Statut:
ppublish
Résumé
The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5'-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.
Identifiants
pubmed: 31863580
pii: 5682902
doi: 10.1093/nar/gkz1170
pmc: PMC7026628
doi:
Substances chimiques
Antiviral Agents
0
Multiprotein Complexes
0
Nucleotides
0
RNA, Viral
0
RNA-Dependent RNA Polymerase
EC 2.7.7.48
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1392-1405Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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