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Journal of Virology, October 2007, p. 11256-11266, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.01028-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue{triangledown}

Jason D. Graci,1 Daniel A. Harki,2,{dagger} Victoria S. Korneeva,1 Jocelyn P. Edathil,2 Kathleen Too,4 David Franco,3,{ddagger} Eric D. Smidansky,1 Aniko V. Paul,3 Blake R. Peterson,2 Daniel M. Brown,4 David Loakes,4 and Craig E. Cameron1*

Department of Biochemistry and Molecular Biology,1 Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802,2 Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York, Stony Brook, New York 11794,3 MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom4

Received 10 May 2007/ Accepted 26 July 2007

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

* Corresponding author. Mailing address: 201 Althouse Lab, University Park, PA 16802. Phone: (814) 865-8703. Fax: (814) 865-7927. E-mail: cec9{at}psu.edu

{triangledown} Published ahead of print on 8 August 2007.

{dagger} Present address: California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena, CA 91125.

{ddagger} Present address: Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016.

Journal of Virology, October 2007, p. 11256-11266, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.01028-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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