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Journal of Virology, May 2003, p. 5152-5166, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5152-5166.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Functional Dissection of a Poliovirus cis-Acting Replication Element [PV-cre(2C)]: Analysis of Single- and Dual-cre Viral Genomes and Proteins That Bind Specifically to PV-cre RNA

Jiang Yin, Aniko V. Paul, Eckard Wimmer, and Elizabeth Rieder*

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

Received 11 September 2002/ Accepted 5 February 2003

The role of the cis replication element (cre) in the 2CATPase coding region of the poliovirus (PV) genome has been studied with a series of mutants derived from either a PV1 full-length genome or a replicon (P/L) containing the firefly luciferase reporter gene in place of the capsid region. Using the P/L replicon we have inserted cre elements at three different locations in the genome including the 5' nontranslated region and within the open reading frame. The successful recovery of replication of a nonviable P/L (A5C) mutant replicon with an artificial cre element as "rescuer," in addition to the results of site-directed mutagenesis and experiments with truncated forms of PV-cre(2C), indicated that (i) the sequence within the upper stem and loop regions contains the minimal cre RNA required for VPg uridylylation in vitro, (ii) the location of the cre RNA in the poliovirus genome is not relevant to RNA infectivity, and (iii) specific binding of 3CDpro to PV-cre(2C) occurs within the upper stem region and probably involves several contact residues. The role of a 14-nucleotide conserved "core" sequence among known cre structures in picornaviruses was examined by site-directed mutagenesis of individual nucleotides. In addition to a conserved AAA (4472 to 4474) triplet previously shown to be the primary RNA template for VPg uridylylation by the PV RNA polymerase 3Dpol (E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10371-10380, 2000), we have now shown that important residues (G4468 and A4481) are contained in a predicted internal bulge at the upper stem-loop of PV-cre(2C). We have further demonstrated that the viral proteins 3CDpro and 3Cpro form stable complexes with a transcript PV-cre(2C) RNA that can be considered critical for VPg uridylylation.

* 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. Phone: (631) 632-8804. Fax: (631) 632-8891. E-mail: ariederrojas{at}notes.cc.sunysb.edu.

Journal of Virology, May 2003, p. 5152-5166, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5152-5166.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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