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

PLoS Pathog. 2014 Jan;10(1):e1003877. doi: 10.1371/journal.ppat.1003877. Epub 2014 Jan 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aedes / virology
  • Amino Acid Substitution
  • Animals
  • Chikungunya virus / enzymology*
  • Chikungunya virus / genetics
  • Chlorocebus aethiops
  • Cricetinae
  • Drosophila melanogaster
  • HeLa Cells
  • Humans
  • Mutation, Missense*
  • RNA Replicase / genetics
  • RNA Replicase / metabolism*
  • Sindbis Virus / enzymology*
  • Sindbis Virus / genetics
  • Vero Cells
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Viral Proteins
  • RNA Replicase

Grant support

This work was supported by European Research Council Starting Grant #242719, French National Grant ANR-09-JCJC-0118-1, and the LabEx IBEID (Integrative Biology of Emerging Infectious Diseases) program. KRG was supported by a stipend from the Pasteur-Paris University (PPU) International PhD Program and by Institut Carnot Pasteur Maladies Infectieuses. Work performed in the laboratory of MCS is supported by French National Grant (ANR-09-JCJC-0045-01), the European Research Council (FP7/2007-2013 ERC 242703), and the LabEx IBEID (Integrative Biology of Emerging Infectious Diseases) program. ABF's contribution was supported by the European Commission Framework Program Seven Award “InfraVec” (project number 228421). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.