The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

Abstract

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D pol at this site excludes its uridylation by the carrier 3D pol . We suggest that VPg at this position is either uridylated by another 3D pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

“…K276 is located at the top of the middle finger and is near the RNA template entry channel, but it does not directly interact with the RNA in any of the picornaviral 3D pol -RNA complexes whose structures have been solved thus far. The structural orientations of the phylogenetically conserved amino acids implicated in the polyadenylation of viral RNA are similar in the atomic structures of 3D pol of enterovirus 71 (35), a group A enterovirus; 3D pol of coxsackievirus B3 (36,37), a group B enterovirus; 3D pol of poliovirus (13,16), a group C enterovirus; 3D pol of rhinovirus type 16 (38,39), species A; 3D pol of rhinovirus type 14 (39), species B; and foot-and-mouth disease virus 3D pol (40,41). These phylogenetically conserved sequences and structures likely contribute to the reiterative transcription of poly(A) and poly(U) sequences during viral RNA replication in these other picornaviruses as well, thereby regulating the lengths of poly(A) at the 3= end of viral RNA.…”

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

“…The polymerase elongation activity assays were performed as described previously (16 Crystallization. Initial crystallization trials of EMCV 3D pol were performed by the sitting-drop vapor diffusion method at both 277 and 293 K in 96-well Greiner plates using a nanoliter-drop crystallization robot (Cartesian Microsys 4000 XL) and the commercial screens: crystal screens 1 and 2 and salt RX (Hampton research).…”

“…To date, the crystal structures of RdRps for six different members of the enterovirus genus and for the aphthovirus FMDV have been reported either isolated or bound to different substrates (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). These enzymes adopt the classical closed right-hand architecture, having finger, palm, and thumb subdomains, with fingertips anchored on the thumb generating an encircled active site.…”

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

“…They have been shown to play a role in key steps of the viral (Gruez et al, 2008; Schein et al, 2006). Although RYMV VPg contains disordered domains in its C-terminal half, a folding into an a-helical conformation can be induced in experimental conditions (Hébrard et al, 2009).…”

“…High-resolution structural data are limited to small VPgs of about 20 residues. The 3D structures of synthetic peptides corresponding to VPgs in complex with viral RNA-dependent RNA polymerase from members of the family Picornaviridae are the only ones available to date (Gruez et al, 2008;Schein et al, 2006). Although RYMV VPg contains disordered domains in its C-terminal half, a folding into an a-helical conformation can be induced in experimental conditions (Hébrard et al, 2009).…”