Mechanistic analysis of RNA-replication elements involved in early stages of chikungunya virus replication.

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Chikungunya virus (CHIKV) is a mosquito-transmitted positive-stranded RNA virus that causes incapacitating chronic joint pain in humans. Having re-emerged as an epidemic in 2004 around the Indian Ocean the virus has been imported into many countries, with >1,135,000 cases reported in the Americas and Europe by 2015. There remains no vaccine or specific antiviral therapy, with development hampered by lack of insight into its biology. Given the scale and expanding geographic distribution of these outbreaks, more research on this virus is urgently needed.In many positive-strand RNA viruses essential mechanisms for regulating of early replication events involve stem-loops within the virus genome. Such structures can act as RNA-replication elements (RRE) and through interaction with host and viral proteins regulate a range of essential processes such as virus translation and genome replication. In preliminary studies we used a combination of structural and reverse genetic analysis to investigate RNA stem-loop elements conserved in protein coding and non-coding regions of the CHIKV genome. In preliminary studies leading to this application we demonstrate that these stem-loops function as RREs during the early stages of CHIKV replication. Silent mutations within each element inhibited CHIKV replication in human cell culture while enhancing it in mosquito cells. In both instances wild-type levels of replication were restored following further mutagenesis to reinstate stem-loop structure. This and further evidence from our preliminary studies lead us to propose that the CHIKV RREs function during early virus replication events, regulating initial translation and genome replication via host specific interaction with trans-activating proteins. I seek support to define host-specific genetic and structural determinants of RRE function, precise roles during early virus replication and specific protein trans-activator interactions. Comparison of results between human and mosquito cell systems, using sub-genomic replicons and infectious virus, will enhance detailed dissection of mechanisms involved. OBJECTIVE-1: Through analysis of engineered mutants across a range of complimentary systems, we will determine precise functional domains within each RRE during early stages of CHIKV replication and generate information on functional local or long-range RNA-RNA/RNA-viral protein interactions. RRE function during CHIKV translation and genome replication will be defined in human and mosquito cell culture.OBJECTIVE-2: Comparative RNA structural analysis will be undertaken using a variety of established and innovative SHAPE methods, producing complimentary quantitative information on RRE structure. Direct insight into the relationship between RNA conformation and function will be provided by comparative analysis of mutants and wild-type under different physiologically relevant conditions and within the replication complex of infected cells.OBJECTIVE-3: Proteomic analysis will be undertaken using a range of in vitro and intracellular techniques, including SILAC mass spectroscopy and PAR-CLIP. Dissecting differences between RRE-proteome interactions in human vs. mosquito cells and native vs. mutated elements will pinpoint interactions relevant to the function of individual RNA structures and stem-loop regions.Through these complimentary in vitro and physiological approaches we will define structural and trans-activating interactions essential to RRE function during CHIKV replication. Roles in translation and genome replication will be defined and through comparison of contrasting host-specific and mutant phenotypes we will dissect functional mechanisms. We expect these results to have direct application in identification of novel therapeutic targets. Furthermore, mechanistic understanding of RRE-mutant attenuation will have direct application in studies towards a genetically stable attenuated vaccine.