Abstract

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.

Highlights

  • During replication, RNA viruses generate approximately 1 error per 104 nucleotides copied, giving rise to an immense population of genetically distinct but closely related variants [1,2,3,4]

  • Like all RNA viruses, during viral replication chikungunya virus (CHIKV) copies its genome using a polymerase that makes an average of one mistake per replication cycle

  • Since Coxsackie virus B3 mutator strains are situated in a structurally analogous area, we hypothesized that this position plays important roles in modulating intrinsic CHIKV RNA-dependent RNA polymerases (RdRp) fidelity [6]

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Summary

Introduction

RNA viruses generate approximately 1 error per 104 nucleotides copied, giving rise to an immense population of genetically distinct but closely related variants [1,2,3,4]. Overall growth and titers of polymerase fidelity variants are not significantly different when grown in isolation in cell culture; for mutators the negative effects of accumulating deleterious mutations are only noticeable after several rounds of replication [6,19]. In recent works, these variants have been useful in exploring how the course of viral infection is affected by either restricted or expanded population diversity [4,10]

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