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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,
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.

Present address: Molecular Pathogenesis Program, Skirball
Institute, New York, NY 10016.
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