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Journal of Virology, October 2008, p. 9400-9408, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00427-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Poliovirus cis-Acting Replication Element-Dependent VPg Uridylylation Lowers the Km of the Initiating Nucleoside Triphosphate for Viral RNA Replication{triangledown}

Benjamin P. Steil1 and David J. Barton1,2*

Department of Microbiology,1 Program in Molecular Biology, University of Colorado Denver, School of Medicine, Aurora, Colorado 800452

Received 26 February 2008/ Accepted 12 July 2008

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5' terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5' end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpUOH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpUOH-independent manner, CRE-dependent VPgpUpUOH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the Km of UTP required for viral RNA replication and that CRE-dependent VPgpUpUOH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.

* Corresponding author. Mailing address: University of Colorado Denver, School of Medicine, Department of Microbiology MS8333, P.O. Box 6511, 12800 East 19th Ave., Aurora, CO 80045. Phone: (303) 724-4215. Fax: (303) 724-4226. E-mail: david.barton{at}uchsc.edu

{triangledown} Published ahead of print on 23 July 2008.

Journal of Virology, October 2008, p. 9400-9408, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00427-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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