Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues

RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis.Financial support was provided by the National Institutes of Health (AI054776 to C.E.C., B.R.P., and J.M.B.), 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 J.P.E. and D.A.H.). We thank Danny Yun and Jeff Baldwin for cloning and purifying RNR using the BL21(DE3)/pET22b expression system. We thank Nora Chapman and Steven Tracy (University of Nebraska Medical Center) for providing plasmids encoding CVB3 cDNA