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.
Highlights
Human enterovirus 71 (EV71), a member of the Enterovirus A (EV A) species belonging to the Enterovirus genus of the Picornaviridae family, is one of the major causative agents of hand, foot and mouth disease (HFMD), and can sometimes cause severe neurological complications such as encephalitis, aseptic meningitis, and acute flaccid paralysis [1,2,3]
The crystal structure was solved at 1.8 Aresolution in space group C2 by molecular replacement using previously reported genotype-B EV71 RNA-dependent RNA polymerase (RdRP) elongation complex (EC) structure as the search model (Table 1) [10]
The RdRP in this EC is structurally consistent with RdRPs in the previously reported enterovirus EC structures [10,43,44,45] with the root-mean-square deviation (RMSD) values in the range of 0.5–1.1 Afor all superimposable ␣carbon atoms
Summary
Human enterovirus 71 (EV71), a member of the Enterovirus A (EV A) species belonging to the Enterovirus genus of the Picornaviridae family, is one of the major causative agents of hand, foot and mouth disease (HFMD), and can sometimes cause severe neurological complications such as encephalitis, aseptic meningitis, and acute flaccid paralysis [1,2,3]. Along with RdRPs from other RNA viruses, the polymerase catalytic core adopts an encircled right hand architecture with palm, fingers and thumb domains surrounding the active site [5,6,7]. The index finger subdomain interacts with the thumb to make the hallmark encirclement distinguished from other classes of processive polymerases [5,6,8]. A collection of viral RdRP structures in complex with RNA or with RNA/NTP further have illustrated the mechanism of the RdRP catalytic mechanisms, in particular, the mechanism of the polymerase nucleotide addition cycle (NAC) with unique features in the pre-catalysis active site closure and post-catalysis translocation events [9,10,11,12,13]
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