The existence of functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognised. In this thesis several computational methods to detect RNA secondary structure in the coding regions of hepatitis C virus (HCV), hepatitis G virus (HGV)/GB virus C (GBV-C) and related viruses have been used. These include thermodynamic prediction of folding free energies (FFEs), evolutionary conservation of minimum energy structures between virus genotypes, suppression of synonymous variability and analysis of covariant and semi covariant substitutions in thermodynamically favoured structures. Each of the predictive methods provided evidence for conserved RNA secondary structure in the core and NS5B encoding regions of HCV and throughout the entire coding region HGV/GBV-C. Positions in the HCV genome with predicted RNA structure localises precisely to regions of marked suppression of variability at synonymous sites, indicating that RNA structure constrains sequence change at what are generally regarded as phenotypically neutral sites. Combining these methods, the computational data obtained in this thesis demonstrates the existence of at least ten conserved stem loop structures within the NS5B encoding region and three in that coding for the core protein in the coding region of HCV. Analysis of the NS5B encoding region and 3’ untranslated region (3’UTR) of HGV/GBV-C indicates an even greater degree of RNA secondary structure. Remarkably, it appears from analysis of FFEs that extensive RNA secondary structure may exist along the entire length of both HCV and HGV/GBV-C genomes, a finding with considerable implications for future functional studies. The existence of predicted RNA structures in the HCV genome was determined using controlled nuclease mapping of RNA transcripts from the core and NS5B regions under conditions which retained potential long-range RNA interactions. The pattern of cleavage sites of nucleases specific for single and double stranded RNA provided strong experimental support for structures previously predicted in this study.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663091 |
| Date | January 2004 |
| Creators | Tuplin, Andrew |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/27558 |
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