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Title: Studies on the importance of the 5' terminal nucleotides of hepatitis C virus in viral RNA replication
Author: Nandasoma, Udvitha Charatha
ISNI:       0000 0004 2714 8160
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2010
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HCV has highly structured RNA elements at the 3' and 5' end of the genome. These conserved regions are not translated and are termed 3' and 5' untranslated regions (UTRs). The terminal and penultimate nucleotides of other flaviviruses are conserved; mutation of these nucleotides compromises RNA replication. The HCV replicon was used to study the effects of mutating the 5' terminal nucleotide on HCV RNA replication. This is a bicistronic RNA molecule containing HCV non structural proteins NS3-58 in conjunction with a selectable neomycin phosphotransferase gene. Replicon transfected cells were selected by adding the antibiotic G418 to culture media. Replicons with a full range of 5' terminal nucleotide mutants were made. A replicon with a purine nucleotide (A or G) could replicate efficiently. A terminal U could support replication at low levels whereas a terminal C could not. Replicons containing a luciferase reporter gene were generated to assess levels of RNA replication at early time points after transfection. These suggested that a terminal A and a terminal G allowed similar levels of RNA replication even at early time points, although a terminal G appeared more efficient at colony formation. In contradiction of previous work, this study found that a terminal A or G was maintained stably in culture. The ability of a replicon with a terminal U to be maintained in culture was dependent on mutation of this nucleotide to A. Studies using the JFH-1 strain of HCV revealed that conventional transcription methods placed non templated nucleotides immediately upstream of the 5'UTR. The native 5' end is restored rapidly in culture and defining this 5'end with a hammerhead ribozyme did not improve rescue efficiency. Analysis of the nucleotides at the +2, +3 and +4 positions of the HCV 5'UTR also suggests that there is some conservation at these sites.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available