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Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Graci, Jason D. and Harki, Daniel A. and Korneeva, Victoria S. and Edathil, Jocelyn P. and Too, Kathleen and Franco, David and Smidansky, Eric D. and Paul, Aniko V. and Peterson, Blake R. and Brown, Daniel M. and Loakes, David and Cameron, Craig E. (2007) Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue. Journal of Virology, 81 (20). pp. 11256-11266. ISSN 0022-538X. http://resolver.caltech.edu/CaltechAUTHORS:GRAjvi07

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Abstract

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 basepairing 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.


Item Type:Article
Additional Information:Copyright © 2007, American Society for Microbiology. Received 10 May 2007/ Accepted 26 July 2007. JVI Accepts, published online ahead of print on 8 August 2007. Financial support was provided by the National Institutes of Health (AI054776 to C.E.C. and B.R.P.; AI015122 to D.F. and A.V.P), the MRC Development Gap Fund (financial assistance to K.T.), and the American Heart Association (Established Investigator award 0340028N to C.E.C. and predoctoral fellowships to D.A.H. and J.P.E.). We thank Christian Castro (Penn State University) for purification of CVB3 3Dpol and Jamie Arnold (Penn State University) for expression and purification of T7 RNA polymerase. We thank Richard R. Drake (Eastern Virginia Medical School) for the gift of HSV-1 TK expression plasmids and purified enzyme. We thank Margaret E. Black (Washington State University) for rabbit HSV-1 TK polyclonal antiserum. We thank Kirk U. Knowlton (University of California, San Diego) for the gift of CVB3 cDNA.
Record Number:CaltechAUTHORS:GRAjvi07
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:GRAjvi07
Alternative URL:http://dx.doi.org/10.1128/JVI.01028-07
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8802
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:18 Sep 2007
Last Modified:26 Dec 2012 09:42

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