Lethal mutagenesis of an RNA plant virus via lethal defection

Ana Grande-Pérez,Isabel Brichette-Mieg,Guillermo Domínguez-Huerta,Axier Pineño-Ramos,Luis Díaz-Martínez

doi:10.1038/s41598-018-19829-6

Abstract

Lethal mutagenesis is an antiviral therapy that relies on increasing the viral mutation rate with mutagenic nucleoside or base analogues. Currently, the molecular mechanisms that lead to virus extinction through enhanced mutagenesis are not fully understood. Increasing experimental evidence supports the lethal defection model of lethal mutagenesis of RNA viruses, where replication-competent-defectors drive infective virus towards extinction. Here, we address lethal mutagenesis in vivo using 5-fluorouracil (5-FU) during the establishment of tobacco mosaic virus (TMV) systemic infections in N. tabacum. The results show that 5-FU decreased the infectivity of TMV without affecting its viral load. Analysis of molecular clones spanning two genomic regions showed an increase of the FU-related base transitions A → G and U → C. Although the mutation frequency or the number of mutations per molecule did not increase, the complexity of the mutant spectra and the distribution of the mutations were altered. Overall, our results suggest that 5-FU antiviral effect on TMV is associated with the perturbation of the mutation-selection balance in the genomic region of the RNA-dependent RNA polymerase (RdRp). Our work supports the lethal defection model for lethal mutagenesis in vivo in a plant RNA virus and opens the way to study lethal mutagens in plant-virus systems.

Highlights

The rapid evolution of viruses is a consequence of their compact genomes, high mutation rates, short replicative cycles and large population sizes that together generate highly variable populations termed viral quasispecies[1,2,3]
According to the suppressor effect of the mutant spectrum observed for lymphocytic choriomeningitis virus (LCMV)[14], it was discovered in persistent infections in cell culture that the transition to extinction with the mutagenic base analogue 5-fluorouracil (5-FU) was characterized by the gradual decrease of virus-specific infectivity, that is, the infectivity per viral genomic RNA molecule, without a concomitant reduction in the number of genomic RNA molecules of the virus[15,16,17]
Lethal mutagenesis using base and nucleoside analogues reduces the infectivity of RNA viruses both in cell culture and in animal models[4]

Summary

Introduction

The rapid evolution of viruses is a consequence of their compact genomes, high mutation rates, short replicative cycles and large population sizes that together generate highly variable populations termed viral quasispecies[1,2,3]. Lethal mutagenesis is founded on a theoretical concept, the error threshold, originally introduced by Manfred Eigen and Peter Schuster in their molecular evolution theory that described the population structure and adaptability of primitive replicons[9,10,11] According to this theory, there is an upper limit in the error rate during the replication of an organism to maintain the genetic information[10]. Increases in the frequency of HCV mutations have been documented[30,34], other studies have not associated the antiviral activity of ribavirin with an increased viral error rate[28,32] Another mutagenic analogue, 5-FU, was able to prevent the in vivo establishment of LCMV persistent infection in mouse[35]. Further studies are needed in vivo to understand the molecular basis of lethal mutagenesis for this antiviral strategy to become a real therapy in the treatment of viral infections

Methods

Results

Conclusion