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Journal of Virology, June 2007, p. 6369-6378, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02252-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Protein-Protein Interactions Critical for Poliovirus Replication: Analysis of 3AB and VPg Binding to the RNA-Dependent RNA Polymerase{triangledown}

Daniel M. Strauss{dagger} and Deborah S. Wuttke*

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215

Received 13 October 2006/ Accepted 26 March 2007

Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 µM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, UCB 215, University of Colorado at Boulder, Boulder, CO 80309-0215. Phone: (303) 492-4576. Fax: (303) 492-5894. E-mail: deborah.wuttke{at}colorado.edu

{triangledown} Published ahead of print on 4 April 2007.

{dagger} Present address: Process Development, Genentech, Inc., One DNA Way, South San Francisco, CA 94080.

Journal of Virology, June 2007, p. 6369-6378, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02252-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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