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Journal of Virology, October 2008, p. 9577-9590, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00631-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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 {triangledown}

Arnaud Gruez ,1,{dagger},{ddagger} Barbara Selisko,1,{dagger}* Michael Roberts,2 Gérard Bricogne,2 Cécile Bussetta,1 Ilham Jabafi,1 Bruno Coutard,1 Armando M. De Palma,3 Johan Neyts,3 and Bruno Canard1*

Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France,1 Global Phasing, Ltd., Sheraton House, Castle Park, Cambridge CB3 0AX, United Kingdom,2 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium3

Received 20 March 2008/ Accepted 10 July 2008

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 3Dpol) 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 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/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.

* Corresponding authors. Mailing address: Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France. Phone: 33 491 82 86 44. Fax: 33 491 82 86 46. E-mail address for B. Selisko: barbara.selisko{at}afmb.univ-mrs.fr. E-mail address for B. Canard: bruno.canard{at}afmb.univ-mrs.fr

{triangledown} Published ahead of print on 16 July 2008.

{dagger} A.G. and B.S. contributed equally to this study.

{ddagger} Present address: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR, 7567, Université Henri Poincaré Nancy I, Vandoeuvre-les-Nancy, France.

Journal of Virology, October 2008, p. 9577-9590, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00631-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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