Viral RNA-dependent RNA polymerase mutants display an altered mutation spectrum resulting in attenuation in both mosquito and vertebrate hosts

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles. Viral replication is then necessary for regeneration of a diverse mutant swarm and this occurs multiple times throughout the viral transmission cycle. 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 virus fidelity results in increased barriers to completing a transmission cycle, a series of mutations in the RNA-dependent RNA-polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for fidelity changes. All putative fidelity mutants were attenuated in both the mosquito and vertebrate hosts, while showing no changes from the wild-type virus 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. The mutants did result in 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 are 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 by using marked 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.