RNA viruses exhibit high mutation rates during genome replication. Nucleotide analogues can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication. They have been explored as antiviral agents acting through lethal mutagenesis. Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, avoiding selection of antiviral-escape mutants. Hepatitis C virus (HCV) is a RNA virus whose infections affect about 180 million people worldwide, and about 75% of newly infected patients progress towards a chronic infection, with a risk of severe liver disease. The main objective of this PhD thesis was to characterize the mechanism of anti-HCV activity produced by ribavirin in hepatoma Huh-7.5 in cell culture. The study led to the observation that guanosine can produce inhibition of HCV progeny production, and that inhibition is also related to a lethal mutagenic effect triggered by this nucleoside. Ribavirin is a recognised mutagenic agent for several other RNA viruses, but it is not clear whether it exerts its anti-HCV activity through mutagenesis or other mechanisms. In the present thesis we provide evidence of a mutagenic activity of ribavirin, documented by statistically significant increases of mutant spectrum complexity (as determinated by mutation frecuency, genetic distances and Shannon entropy), and a mutational bias in favor of G→A and C→U transitions. Both molecular cloning and Sanger sequencing, and ultra-deep pyrosequencing have been used for the analysis of HCV populations. Ribavirin treatment resulted in nucleotide imbalances (a reduction of intracellular GTP and an increase of UTP, ATP and CTP). Control experiments using mycophenolic acid and guanosine indicated that GTP depletion cannot explain ribavirin mutagenesis. Moreover, HCV extinction by ribavirin, but not by the non-mutagenic HCV inhibitor mycophenolic acid, occurred with decreases of specific infectivity, a feature typical of lethal mutagenesis. Thus, at least part of the antiviral activity of ribavirin on HCV in Huh-7.5 cells is exerted via lethal mutagenesis. Unexpectedly, guanosine which partially counteracted the inhibitory activity of ribavirin, inhibited HCV progeny production. We studied the effect of high concentrations of guanosine and other nucleotides on HCV and Huh-7.5 cells. Guanosine, but no the other nucleosides, produces a general decrease of the NTP/NDP ratios. Guanosine exerts its anti- HCV activity through mutagenesis, with a significant increase of the proportion of deletions and insertions, accompanied of a decrease of specific infectivity. This antiviral activity is not observed with other RNA viruses, and it constitutes the first example of a metabolite-induced lethal mutagenesis. The results open the way to study means to increase the efficacy of lethal mutagenesisbased treatments for HCV infections.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-10-2014