Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance

Abstract

<jats:p>It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for ϕX174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.</jats:p>

Trends in antiviral strategies

“…A theoretical model developed by S. Manrubia predicted that such defective genomes that were termed defectors were required to achieve LCMV extinction with a limited mutagenic intensity by FU (Grande-P erez et al, 2005b). The proposal that loss of viral infectivity is due to the action of defective genomes produced by the mutagenic agent is termed the lethal defection model of virus extinction, and it is consistent with several observations on the extinction of other viruses (reviewed in Domingo et al, 2012). A diagnostic test is that loss of infectivity precedes loss of viral RNA ( Fig.…”

Trends in Antiviral Strategies

“…A decade later the first ribavirin-resistant mutants of poliovirus selected in the laboratory (Pfeiffer and Kirkegaard, 2003), and of HCV from patients under ribavirin monotherapy were described (Young et al, 2003). Subsequent work has characterized viral mutants resistant to mutagenic agents, particularly ribavirin, FU, and 5-AZA-C (Pfeiffer and Kirkegaard, 2005b;Sierra et al, 2007;Arias et al, 2008;Agudo et al, 2010;Levi et al, 2010;Arribas et al, 2011;Feigelstock et al, 2011;Domingo-Calap et al, 2012;Sadeghipour et al, 2013;Zeng et al, 2013Zeng et al, , 2014; among other studies). The major overall conclusion of these investigations is that viruses can develop resistance to mutagenic nucleotide analogs as they do to nonmutagenic inhibitors.…”

Genomic mutation rates that neutralize adaptive evolution and natural selection

“…A large increase in the mutation rate can create strong selection pressure for a lowered mutation rate, and a reduction in the mutation rate may thus evolve in short order. Our own work with a mutator strain of Escherichia coli and a nucleoside analogue mutagen, together with several previous mutagenesis studies using different viral systems, shows that resistance to mutagens at high doses can evolve rapidly and through a number of different mechanisms [76][77][78][79][80][81][82]. If one could increase the mutation rate to a level that is high enough to cause extinction, but not too high, selection for resistance could, in principle, be reduced considerably and the evolution of resistance might be prevented.…”