The existence and 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 of HGV/GBV-C.Positions in the HCV genome with predicted RNA structure localise 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 coding region and three in that coding for the core
protein both within the coding region of HCV. Analysis of the NS5B coding 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 the
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. Electron microscopy was also used to directly visualise the RNA folding
structure of HGV/GBV-C and provided some evidence for at least four structures
within the NS5B coding region and long range RNA folding across the length of the
virus genome.The degree of structural conservation between diverse HCV and HGV/GBV-C
genotypes and related viruses suggests roles in virus replication, and/or RNA
packaging for the discrete structures identified in this thesis. Whilst this role and that
of the genome wide structure identified is currently not understood the structures
predicted in this work are providing a starting point for such functional studies using
the HCV replicon
