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J Virol, February 1998, p. 1297-1307, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Protein Linkage Map of the P2 Nonstructural Proteins of Poliovirus

Andrea Cuconati, Wenkai Xiang,dagger Frederick Lahser, Thomas Pfister, and Eckard Wimmer*

Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794

Received 2 July 1997/Accepted 5 November 1997

The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2Apro, 2BC, 2C, and 2B. Specifically, 2Apro can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. trans-dominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.

* 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, New York, NY 10016.




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