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Journal of Virology, August 2006, p. 7405-7415, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.02533-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

Oliver C. Richards,1 Jeannie F. Spagnolo,2 John M. Lyle,2,{dagger} Susan E. Vleck,2 Robert D. Kuchta,1 and Karla Kirkegaard2*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado,1 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California2

Received 4 December 2005/ Accepted 4 May 2006

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305-5402. Phone: (650) 498-7075. Fax: (650) 498-7147. E-mail: karlak{at}stanford.edu.

{dagger} Present address: Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025.

Journal of Virology, August 2006, p. 7405-7415, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.02533-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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