Molecular Basis of Fitness Loss and Fitness Recovery in Vesicular Stomatitis Virus

Viral populations subjected to repeated genetic bottleneck accumulate deleterious mutations in a process known as Muller's ratchet. Asexual viruses, such as vesicular stomatitis virus (VSV) can recover from Muller's ratchet by replication with large effective population sizes. However, mutants with a history of bottleneck transmissions often show decreased adaptability when compared to non-bottlenecked populations. We have generated a collection of bottlenecked mutants and allowed them to recover by large population passages. We have characterized fitness changes and the complete genomes of these strains. Mutations accumulated during the operation of Muller's ratchet led to the identification of two potential mutational hot spots in the VSV genome. As in other viral systems, transitions were more common than transversions. Both back mutation and compensatory mutations contributed to recovery, although a significant level of fitness increase was observed in nine of the 13 bottlenecked strains with no obvious changes in the consensus sequence. Additional replication of three strains resulted in the fixation of single point mutations. Only two mutations previously found in non-bottlenecked, high-fitness populations that had been adapting to the same environment were identified in the recovered strains.

Introduction

Repeated bottlenecks during the evolution of a population lead to the accumulation of deleterious mutations and overall fitness losses in a process known as Muller's ratchet.¹ Sexual populations can recover from Muller's ratchet by recombination, but asexual organisms need to rely on mutation for fitness recovery. RNA viruses have been excellent models to study the operation of Muller's ratchet,2, 3, 4, 5, 6 and the mechanisms underlying fitness loss.⁷ Fitness recovery in sexual viruses has been studied in foot-and-mouth disease virus (FMDV) and phage ϕ6.7, 8 FMDV is a positive stranded RNA virus, with the ability to undergo homologous recombination,9, 10 but the role of recombination during fitness recovery in this virus has not been studied. In contrast ϕ6 is a segmented RNA virus, so genetic exchange occurs by reassortment of segments during coinfection, and this contributes to fitness recovery.11, 12 In spite of this ability, mutation is the major source of genetic variation leading to fitness recovery in both viral species.

Vesicular stomatitis virus (VSV) is a negative stranded RNA virus, and homologous recombination does not occur to any significant extent,13, 14 making it effectively asexual. VSV belongs to the Rhabdoviridae family. The genome is a single molecule of negative stranded RNA that is 11,161 nt long and encodes five genes. The RNA is found in complexes that incorporate three viral proteins (N, P and L), the nucleocapsid. These are attached to an external membrane envelope by the M protein, and the external G protein allows receptor-mediated entrance to the cell. Extensive work has been done to characterize fitness changes during Muller's ratchet in VSV populations.3, 15, 16, 17, 18 In addition, mutants with a history of genetic bottleneck typically have an impaired adaptability compared to strains that have been kept at relatively large population sizes.19, 20, 21 Here, we present the genetic characterization of a collection of mutants generated by repeated plaque-to-plaque passages, as well as populations evolved from these strains under strict positive selection. Recovery from the accumulation of deleterious mutations took place both by compensation and back mutation, but a large fraction of recovered populations showed significant fitness increases in the absence of mutation accumulation.