RNA viruses replicate with a very high error rate and give rise to heterogeneous, highly plastic populations able to adapt very rapidly to changing environments. Viral diseases are thus difficult to control because of the appearance of drug-resistant mutants, and it becomes essential to seek mechanisms able to force the extinction of the quasispecies before adaptation emerges. An alternative to the use of conventional drugs consists in increasing the replication error rate through the use of mutagens. Here, we report about persistent infections of lymphocytic choriomeningitis virus treated with fluorouracil, where a progressive debilitation of infectivity leading to eventual extinction occurs. The transition to extinction is accompanied by the production of large amounts of RNA, indicating that the replicative ability of the quasispecies is not strongly impaired by the mutagen. By means of experimental and theoretical approaches, we propose that a fraction of the RNA molecules synthesized can behave as a defective subpopulation able to drive the viable class extinct. Our results lead to the identification of two extinction pathways, one at high amounts of mutagen, where the quasispecies completely loses its ability to infect and replicate, and a second one, at lower amounts of mutagen, where replication continues while the infective class gets extinct because of the action of defectors. The results bear on a potential application of increased mutagenesis as an antiviral strategy in that low doses of a mutagenic agent may suffice to drive persistent virus to extinction.

Studies with several RNA viruses have shown that enhanced mutagenesis resulted in decreases of infectivity or virus extinction, as predicted from virus entry into error catastrophe. Here we report that lymphocytic choriomeningitis virus, the prototype arenavirus, is extremely susceptible to extinction mutagenesis by the base analog 5-fluorouracil. Virus elimination was preceded by increases in complexity of the mutant spectra of treated populations. However, careful molecular comparison of the mutant spectra of several genomic segments suggests that the largest increases in mutation frequency do not predict virus extinction. Highly mutated viral genomes have escaped detection presumably because lymphocytic choriomeningitis virus replicates at or near the error threshold, and genomes in the transition toward error catastrophe may have an extremely short half-life and escape detection with state-of-the-art cloning and sequencing technologies.

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.

Enhanced mutagenesis may result in RNA virus extinction, but the molecular events underlying this process are not well understood. Here we show that 5-fluorouracil (FU)-induced mutagenesis of the arenavirus lymphocytic choriomeningitis virus (LCMV) resulted in preextinction populations whose consensus genomic nucleotide sequence remained unaltered. Furthermore, fitness recovery passages in the absence of FU, or alternate virus passages in the presence and absence of FU, led to profound differences in the capacity of LCMV to produce progeny, without modification of the consensus genomic sequence. Molecular genetic analysis failed to produce evidence of hypermutated LCMV genomes. The results suggest that low-level mutagenesis to enrich the viral population with defector, interfering genomes harboring limited numbers of mutations may mediate the loss of infectivity that accompanies viral extinction.

Nanoviruses are multipartite single-stranded DNA (ssDNA) plant viruses that cause important diseases of leguminous crops and banana. Little has been known about the variability and molecular evolution of these viruses. Here we report on the variability of faba bean necrotic stunt virus (FBNSV), a nanovirus from Ethiopia. We found mutation frequencies of 7.52 × 10 ⁻⁴ substitutions per nucleotide in a field population of the virus and 5.07 × 10 ⁻⁴ substitutions per nucleotide in a laboratory-maintained population derived thereof. Based on virus propagation for a period of more than 2 years, we determined a nucleotide substitution rate of 1.78 × 10 ⁻³ substitutions per nucleotide per year. This high molecular evolution rate places FBNSV, as a representative of the family Nanoviridae , among the fastest-evolving ssDNA viruses infecting plants or vertebrates.