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

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.

Chikungunya virus (CHIKV) is a mosquito-transmitted 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 preliminary studies we used a combination of structural and reverse genetic analysis to investigate stem-loop RNA replication elements (RREs) conserved in coding and non-coding regions of the CHIKV genome. Results presented in the 'Case For Support' demonstrate that each of the individual RREs functions during the early stages of CHIKV replication, through a host dependent mechanism. This application seeks support to define genetic, structural and trans-activating interactions essential to RRE function during CHIKV replication, through complimentary in vitro and physiological approaches. Roles in translation and genome replication will be defined by reverse genetic analyses of RRE mutants and through comparison of contrasting host-specific vs. 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, which we would aim to exploit through future studies involving both academic and industrial partners.

Through a range of academic, economic and societal impacts, three distinct groups will benefit from the proposed research.

1) Lack of understanding regarding the biology of chikungunya virus (CHIKV) replication in mammalian and mosquito hosts has hindered both vaccine development and that of antiviral therapies. Consequently, given the expanding scale and geographic distribution of CHIKV outbreaks (across tropical/subtropical regions into temperate latitudes including Western Europe and the USA) more research is urgently needed. I expect outcomes of this proposal to translate into a number of important positive impacts for CHIKV biomedical research in the UK and worldwide. In particular an improved understanding of how RNA replication element (RRE)-protein interactions control CHIKV replication will be invaluable for identifying novel targets for therapeutic intervention. Furthermore, as demonstrated for poliovirus, a mechanistic understanding or RRE-mutant attenuation will have direct application in studies towards a stably attenuated vaccine. Consequently, completion of the proposed study will have a substantial impact on biomedical research aimed at preventing or controlling this significant human pathogen. In the longer term, as well as the health benefits, there are clear wealth implications both for the company developing a successful vaccine or therapy and the nation able to derive tax revenue from sales.

2) Enhancing our understanding of how replication and translation is controlled during CHIKV infection will provide academic impact benefits to UK and international researchers interested in furthering our understanding of CHIKV (and related alphavirus) biology and its interaction with host factors, such as innate immune responses. Furthermore, advancing our knowledge of how structured-RNA elements function and interact within the physiological intracellular environment will be of great interest to the wider virology and general scientific community, researching fundamental biological mechanisms. As part of the proposed project we intend to utilise a highly innovative methodology, for intracellular mapping of structured-RNA during active virus replication. This technique will have direct cross-disciplinary application across the wider virology and cell biology fields.

3) The UK's international strength and economic competitiveness within biomedical research and the pharmaceutical sector is reliant on a pool of highly trained researchers. Both the PDRA and technician involved in prosecuting the proposed project will receive first class training across a range of modern virology, molecular/cell biology and bioinformatic techniques, as well as safe working under BSL-3 containment; providing an excellent base for a successful research career within either academic or commercial biomedical research. Increasing the pool of such highly trained biomedical scientists will contribute positively to increased capacity in the wider UK research base and more specifically within the internationally competitive field of arbovirus virology. These benefits will extend to graduate students, undergraduates and visitors who will also benefit from training received in my laboratory. Researchers will gain training and experience in a range of skills transferable to employment in non-research or non-science sectors, including analytical thinking, data analysis, computational skills, public speaking and academic writing. Skills essential for enhancing the UK's success as a modern knowledge based economy.