No abstract

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.Natural nucleotides exist as tautomers in solution, and tautomerization of the nucleobases in DNA has been recognized as a likely mutagenic mechanism ever since the double-helical structure of the DNA molecule was first deduced by Watson and Crick (33,36). The keto (for G and U) and amino (for A and C) tautomers of the natural nucleotides are the predominant species with tautomeric constants (K T ) on the order of 10 5 . However, tautomeric conversion to the rare enol or imino forms of the nucleobases can lead to altered hydrogen bonding specificity and thus mutagenesis through noncanonical basepairing interactions. This has become known as the "rare tautomer" hypothesis of mutation (19,29).The tautomerization of bases to yield ambiguous base-pairing properties has also been exploited in the design of novel nucleoside drugs, including 5-hydroxy-2Ј-deoxycytidine (22) and 5-aza-5,6-dihydro-2Ј-deoxycytidine (KP-1212) (18, 25) (Fig. 1A). However, attempts to design clinically useful antiviral compounds around this premise have met with only limited success (13,16,18,25).Nucleobases exhibiting multiple conformations due to rotation or tautomerization have received attention as potential antiviral agents acting through lethal mutagenesis (11,22). Since the discovery that ribavirin (Fig. 1B) can act as a lethal mutagen (7), likely through rotation of the exocyclic carboxamide moiety, the concept of lethal mutagenesis as an antiviral strategy has received considerable attention (5,11,12).Recently, we have demonstrated that the ribonucleoside analogue rP (Fig. 1C) can act as a potent mutagen of poliovirus (PV) in vitro (13). Tautomerization of the nucleobas...

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.

A number of aromatic universal base analogues have been described in the literature, but most are non-hydrogen bonding. We have examined the effect of introducing hydrogen bonding carboxamide groups onto the pyrrole ring of 5-nitroindole. The modified analogues retain universal base features, but there are no overall effects on duplex stability. This leads to the suggestion that the nitro group is within the hydrogen bonding face of the duplex, and the hydrogen bonding carboxamide group is in the duplex major groove.