Abstract
The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.
Highlights
RNA viruses comprise a diverse group of viruses, which exhibit high genome plasticity due to a high rate of mutation
RNA viruses replicate with a high mutation rate, giving them the ability to generate mutations that might be beneficial under different selection pressures
Using a model system of Venezuelan equine encephalitis virus (VEEV) we investigated the effect of perturbing the rate of mutations in both a mosquito and vertebrate host
Summary
RNA viruses comprise a diverse group of viruses, which exhibit high genome plasticity due to a high rate of mutation This results in the generation of a closely related cloud of viral variants known as a ‘quasispecies’ or viral swarm [1]. Rather than producing perfect copies of the genome, the RdRp randomly incorporates incorrect nucleotide bases along the genome, generating a cloud of viral genomes that contain one or two mutations in each genome [2]. These variants are believed to collectively contribute to an interactive population that together create the viral phenotype. For arboviruses (arthropod-borne viruses) creating a diverse quasispecies is hypothesized to be of utmost importance, as two diverse species, a vertebrate host and invertebrate vector, must become infected to complete a transmission cycle, and recent work has demonstrated that diversity is important in successful completion of a transmission cycle [4,5,6,7]
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