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Title: The role of RNA structure in Chikungunya virus early replication events
Author: Kendall, Catherine Theresa
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Chikungunya virus (CHIKV) is a pathogenic, single-stranded, positive-sense RNA virus transmitted to humans by Aedes spp. mosquitoes. After decades of low-level endemic circulation, CHIKV has re-emerged to establish local transmission on five continents, infecting upwards of 3,000,000 people. There are no currently available vaccines or direct-acting anti-viral therapeutic agents. A greater understanding of the CHIKV replication cycle is essential, as much of what is known about the replication cycle is assumed from studies of related but divergent viruses, which have provided conflicting reports. Preliminary work carried out by A. Tuplin (University of Leeds) suggested a highly ordered structured region at the 5′ end of the CHIKV genome, spanning ~300 nt including the 5′ untranslated region (UTR) and the 5′ coding sequence of nsp1. The aim of this project was to determine the phenotypic importance of secondary structure in this region for the CHIKV lifecycle in human and mosquito cells at multiple stages of viral replication and to investigate the sequence and structure requirements for functional interactions. This study represents the first investigation of functional elements within the 5′ UTR and adjacent nsP1-coding region in CHIKV. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, the wild-type secondary structure of the 5′ 300 nt of the CHIKV genome was found to be essential for genome replication in human- and Ae. albopictus-derived cells. Six RNA stem-loops were determined to individually enhance CHIKV genome replication - including novel structures analysed for the first time in this study. Comparative analysis in human and mosquito-derived cell lines revealed that the novel stem-loop SL47 in the 5′ UTR functions in a host-independent manner while stem-loops in nsp1 function in a host-dependent manner. Stem-loops were demonstrated to function within the positive-strand genomic RNA, via predominantly structure-dependent mechanisms. Furthermore, single-host passaging studies suggested strong selection pressure to regenerate secondary structures and highlighted potential differences in translational recoding between host species. Finally, the potential for tertiary structure formation was explored. In addition to furthering knowledge of fundamental aspects of the molecular virology of this important human pathogen, this study will inform rational design of a genetically stable attenuated vaccine.
Supervisor: Tuplin, Andrew ; Stonehouse, Nicola J. Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available