A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Shi W,Ye HQ,Deng CL,Li R,Zhang B,Gong P

doi

10.1093/nar/gkz1170

subject

Has Abstract

pub_date

2020-02-20 00:00:00

pages

1392-1405

issue

3

eissn

0305-1048

issn

1362-4962

pii

5682902

journal_volume

48

pub_type

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