Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity. Author Summary Chikungunya (CHIKV) is a re-emerging mosquito-borne virus that constitutes a major and growing human health burden. Like all RNA viruses, during viral replication CHIKV copies its genome using a polymerase that makes an average of one mistake per replication cycle. Therefore, a single virus generates millions of viral progeny that carry a multitude of distinct mutations in their genomes. In this study, we isolated CHIKV mutators (strains that make more errors than the wildtype virus), to study how higher mutation rates affect fitness in arthropod-borne viruses (arboviruses). CHIKV mutators have reduced virulence in mice and severe replication defects in Aedes mosquito cells. However, these replication defects result in selective pressure for reversion of mutators to a wildtype polymerase in mosquito hosts. To examine how mutators would behave in an insect model in absence of this genetic instability, we isolated mutators of a related virus, Sindbis virus (SINV). SINV mutators had no replication defect in fruit fly (Drosophila) cells, and a SINV mutator strain was stable and attenuated in fruit flies. This work shows proof of principle that arbovirus mutators can exhibit attenuation in both mammalian and insect hosts, and may remain a viable vaccine strategy.