Structural and phenotypic analysis of Chikungunya Virus RNA structures during viral genome replication and translation

Chikungunya virus (CHIKV) is a pathogenic, positive-sense RNA virus of the Alphavirus genus, which causes fever and debilitating joint pain in humans. CHIKV is transmitted by Aedes mosquitoes and is currently re-emerging in the absence of approved vaccines or antiviral therapies. One approach to vaccine production is to attenuate CHIKV replication through disruption of genomic RNA secondary structures. We have mapped the RNA structure of the 5’ region of the CHIKV genome using selective 2’-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. We have identified five highly-conserved RNA structures within the nsP1-coding region of ORF-1 and, using a reverse genetics approach, determined their impact on virus replication in infectious virus and luciferase-reporter subgenomic replicon systems. Our results suggest that RNA structures within the nsP1-coding region are required for efficient CHIKV genome replication in mammalian and mosquito cells, potentially via vertebrate/invertebrate host-specific mechanisms. For example, disruption of one of the stem-loops inhibits CHIKV replication in mammalian cell lines, while having no significant effect in mosquito cells. Restoration of the structure via compensatory silent mutations restores replication – indicating RNA structure-dependent enhancement of CHIKV replication. Conversely, disruption of an adjacent stem-loop inhibits CHIKV replication in mosquito cells but not in mammalian cell lines. Our structural data also suggests that higher-order interactions within this region impact CHIKV replication. As arboviruses continue to re-emerge, it is critical that we improve our understanding of their replication cycles. RNA structures may constitute novel targets for vaccine attenuation or drug design.