Cross-utilisation of template RNAs by alphavirus replicases

Alphaviruses are positive-strand RNA viruses, most of which use mosquitoes to spread between vertebrate hosts; many are human pathogens with potentially severe medical consequences. Some alphavirus species are believed to have resulted from the recombination between different members of the genus and there is evidence of movement of alphaviruses between continents. Here, a novel assay uncoupling viral replicase and template RNA production was developed and used to analyse cross-utilization of alphavirus template RNAs. We observed that replicases of closely related alphaviruses belonging to the Semliki Forest virus complex can generally use each other’s template RNAs as well as those of distantly related outgroup viruses. In contrast, replicases of outgroup viruses clearly preferred homologous template RNAs. These trends were observed in both mammalian and mosquito cells, with template preferences generally more pronounced in mosquito cells. Interestingly, the template RNA of the mosquito-specific Eilat virus was efficiently used by other alphavirus replicases while Eilat replicase could not use heterologous templates. Determinants for template selectivity were mapped to the beginning of the RNA genome and template recognition was more likely based on the recognition of RNA sequences than recognition of structural elements formed by the RNAs.