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J Virol, August 1998, p. 6732-6741, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

Wenkai Xiang,1,dagger Andrea Cuconati,1,Dagger Debra Hope,2 Karla Kirkegaard,3 and Eckard Wimmer1,*

Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-52221; Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-03472; and Department of Microbiology and Immunology, Stanford University, Stanford, California 94365-54023

Received 5 February 1998/Accepted 6 May 1998

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (516) 632-8787. Fax: (516) 632-8891. E-mail: wimmer{at}asterix.bio.sunysb.edu.

dagger Present address: Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.

Dagger Present address: Center for Advanced Biotechnology and Medicine, Department of Biological Sciences, Rutgers University, Piscataway, NJ 08854.

J Virol, August 1998, p. 6732-6741, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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