Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

Abstract

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.

Increased Replicative Fitness Can Lead to Decreased Drug Sensitivity of Hepatitis C Virus

“…Transitions amounted to 75% to 77% of the total number of mutations, and the ratio [(G¡A)ϩ(C¡U)]/[(A¡G)ϩ(U¡C)] was maintained in the range of 0.21 to 0.69 (see Fig. S3), as expected for HCV not subjected to a mutagenic agent (53). The amino acid substitutions deduced from the NS5A mutant spectra of HCV p45 and HCV p100 did not reveal any substitutions (F28S, L31M, C92R, and Y93H) currently catalogued as conferring resistance to daclatasvir (67-73) (see Tables S7 and S8).…”

“…The partial resistance extends also to ribavirin, which acts as an HCV mutagen in our experimental system (53). Despite an expansion of the mutant spectrum complexity during HCV passage in Huh-7.5 cells (Tables 2 and 3), the drug resistance phenotype was not due to the presence of any inhibitor resistance mutations that might have accumulated in the expanded mutant spectrum of HCV.…”

“…telaprevir and daclatasvir in HCV p100. The variation of statistical significance with MOI in the case of ribavirin is not easy to interpret since under our infection conditions ribavirin acts as both an inhibitor and a mutagen (53). Resistance to telaprevir was examined with more detail.…”

“…Nucleotide sequences of genomic HCV RNA were determined on the two strands of an amplified cDNA copy (45,52). To evaluate the complexity of mutant spectra, HCV RNA was extracted as described above and subjected to RT-PCR to amplify the NS5A-coding regions as previously described (53). To ensure an excess of template in the RT-PCR amplifications for quasispecies analysis, and to avoid complexity biases due to redundant amplifications of the same initial RNA templates, amplifications were carried out with template preparations diluted 1:10, 1:100, and 1:1,000; only when the template diluted 1:100 produced a visible DNA band was molecular cloning pursued using the DNA amplified from the undiluted template.…”

“…Negative controls (without template RNA) were run in parallel to ascertain the absence of contamination with undesired templates. Procedures for the data analysis have been previously described (53). The genomic nucleotide sequences of the HCV described in the present study have been deposited in GenBank (see below).…”