Rare haplotype load as marker for lethal mutagenesis.

Josep Gregori,Celia Perales,Isabel Gallego,Juan Ignacio Esteban,Mercedes Guerrero-Murillo,Esteban Domingo,Josep Quer,María Eugenia Soria,Jean-Pierre Vartanian

doi:10.1371/journal.pone.0204877

Abstract

RNA viruses replicate with a template-copying fidelity, which lies close to an extinction threshold. Increases of mutation rate by nucleotide analogues can drive viruses towards extinction. This transition is the basis of an antiviral strategy termed lethal mutagenesis. We have introduced a new diversity index, the rare haplotype load (RHL), to describe NS5B (polymerase) mutant spectra of hepatitis C virus (HCV) populations passaged in absence or presence of the mutagenic agents favipiravir or ribavirin. The increase in RHL is more prominent in mutant spectra whose expansions were due to nucleotide analogues than to multiple passages in absence of mutagens. Statistical tests for paired mutagenized versus non-mutagenized samples with 14 diversity indices show that RHL provides consistently the highest standardized effect of mutagenic treatment difference for ribavirin and favipiravir. The results indicate that the enrichment of viral quasispecies in very low frequency minority genomes can serve as a robust marker for lethal mutagenesis. The diagnostic value of RHL from deep sequencing data is relevant to experimental studies on enhanced mutagenesis of viruses, and to pharmacological evaluations of inhibitors suspected to have a mutagenic activity.

Highlights

The mutant spectra of RNA viruses are a reflection of their evolutionary history, as well as important determinants of virus adaptability
Infectious progeny levels were those expected from previous quantifications of inhibition of hepatitis C virus (HCV) p0 by favipiravir [19] and ribavirin [34]; the sustained HCV p100 and HCV p200 production in the presence of the drugs is expected from the fitness-associated HCV resistance to antiviral agents [31, 32, 35] (Fig 1B)
Intracellular viral RNA was sequenced in MiSeq with 2x300 mode with v3 chemistry, and fastq files were analyzed as previously described [27, 29] to obtain forward and reverse consensus haplotypes with abundances not below 0.1%, median coverage 147,000 reads, interquartile range (IQR) 75570–226100

Summary

Introduction

The mutant spectra of RNA viruses are a reflection of their evolutionary history, as well as important determinants of virus adaptability. Concerning control of viral diseases, mutant spectrum dynamics is an obstacle for the efficacy of therapeutic interventions due to selection of treatment-escape viral mutants. The antiviral agents to combat RNA viruses include those directed to specific viral targets [direct-acting antiviral agents (DAAs)], and those that inhibit cellular functions needed for the completion of the virus life cycle. The viral RNA-dependent RNA polymerase (RdRp) is the target of several effective antiviral agents. Notably base or nucleoside analogues, are intracellularly converted into their active nucleotide.

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Results

Conclusion