Unravelling a co-nsP-iracy : investigating the functional characteristics of Chikungunya virus non-structural protein 3

Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by the Aedes species of mosquito. Infection with CHIKV causes chikungunya fever, which can lead to debilitating chronic joint disease. Despite the rising potential as a threat to global health, no effective vaccine nor antiviral agents for prophylaxis or treatment are available. The CHIKV non-structural protein 3 (nsP3) is essential to the virus lifecycle and is required for genome replication. However, to date, the exact role of this protein remains unclear. In in vitro studies, nsP3 has been shown to bind promiscuously to RNA but the precise nature of such interactions in vivo has not been explored. To investigate this, individual nucleotide UV-crosslinking and immunoprecipitation (iCLIP) combined with next generation sequencing were employed. This analysis revealed that nsP3 preferentially binds to the CHIKV genome, over host RNA. The nature of nsP3 binding to viral RNA also appeared stochastic. Further bioinformatics analyses were then applied to assess how nsP3 is able to recognise target RNA for binding. However, neither a consensus binding motif nor specific preferences for secondary RNA structures were identified, suggesting that these may not be key drivers of RNA binding recognition for this protein. In parallel, this project also aimed to investigate a polyproline motif located in the C-terminal HVD of nsP3 which is important for RNA synthesis and interactions with host amphiphysin in multiple alphaviruses but the relevance in CHIKV has not been fully investigated. To address this, a panel of mutations targeting conserved residues of the motif were generated in both CHIKV sub-genomic replicon and full length infectious virus. Most of the mutants were able to replicate and generate infectious virus in the respective systems, except one mutant (P398A/P401A) which was able to replicate in the subgenomic system but presented reduced replication and a complete inability to produce infectious virus. Further analysis revealed that the mutation responsible for the phenotype was not the intended mutation in the polyproline motif, but instead located to the 3' UTR, which is essential for negative strand synthesis and subsequent genome replication. The results indicate that the polyproline motif is not absolutely essential for the CHIKV lifecycle.