Untangling the impact of mutations in the coding region of chikungunya virus nsP1

Chikungunya virus (CHIKV) is a single-stranded, positive-sense RNA virus of the alphavirus genus and is the known causative agent of chikungunya fever. CHIKV is an arbovirus and transmitted between humans and Aedes spp. mosquitoes. Increasing global temperatures has resulted in the expansion of the mosquitoes geographical range. As a consequence the epidemic incident rate of CHIKV is increasing and it is now recognised as a potential threat to global health. There are currently no approved specific therapeutics or vaccines against CHIKV, so a greater understanding into the CHIKV lifecycle is essential. Preliminary data from our group demonstrated that the presence of a second methionine (M24) in the non-structural protein 1 (nsP1) coding region enhanced CHIKV replication and hypothesised that this residue functioned as an alternative start codon to initiate translation of truncated nsP1. A panel of mutants were introduced at M24 in the full-length infectious clone, sub-genomic replicon and trans-replicase assay constructs. Attenuated virus replication was observed across the mutant panel in both mammalian and mosquito cells. Further experiments demonstrated that mutations did not inhibit replication in the context of nsP1 functioning in the replicase complex, but rather in the context of the RNA template. Previous studies have clearly demonstrated the importance of RNA structures within the 5′ conserved sequence element. To understand the impact to RNA secondary structures across the mutant panel, selective 2′ hydroxyl acetylation analysed by primer extension (SHAPE) was used to inform the modelling of RNA structures within the 5′ region of CHIKV. The SHAPE-constrained minimum free energy models confirmed that these structures were disrupted to varying extents across the mutant panel, except M24S which had high structural similarity with wild-type CHIKV. In order to distinguish a specific mechanism for this disrupted region, M24A was passaged for 10 rounds in human and mosquito cells, and alternately between the two cell lines. Extracted cellular RNA was analysed by Next Generation Sequencing, and substitutions were observed at nucleotide C147U following passaging in human cells and alternate passaging between human and mosquito cell lines. Pseudo-escape mutations were observed at nucleotide A77G, which caused an amino acid change of the ORF-1 start codon to a valine. Attempts to confirm whether these mutations were sufficient to restore virus replication to wild-type levels remained inconclusive. However, it was demonstrated that these mutations were sufficient to restore wild-type base pairing in RNA structures. In parallel, this project aimed to determine the molecular structure of CHIKV nsP1 through X-ray crystallography. Recombinant full-length CHIKV nsP1 was successfully expressed and purified through a bacterial expression system and nickle affinity chromatography however contamination products were consistently present following size exclusion chromatography, and therefore the protein was not taken forward for X-ray crystallography trials.