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Journal of Virology, April 2003, p. 4739-4750, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4739-4750.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis

Kenneth E. Murray1 and David J. Barton1,2*

Department of Microbiology,1 Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 802622

Received 29 August 2002/ Accepted 16 January 2003

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

* Corresponding author. Mailing address: Department of Microbiology, Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Phone: (303) 315-5164. Fax: (303) 315-6785. E-mail: david.barton{at}uchsc.edu.

Journal of Virology, April 2003, p. 4739-4750, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4739-4750.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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