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Title: Investigation of the role of viral proteins in human immunodeficiency virus type-1 RNA translation
Author: Groom, H. C. T.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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Our laboratory has identified an additional Rev binding site in the 5’ UTR of HIV-1 RNA. This site is referred to as Loop A and is found on Stem Loop 1 of the packaging signal in the 5’ untranslated region. We tested the effect of Rev on translation of HIV-1 RNA to dissect the roles of the RRE and Loop A. In vitro, Rev stimulates translation of HIV-1 RNAs at intermediate concentrations and inhibits translation non-specifically at high concentrations. Stimulation appears to be dependent on the presence of Loop A rather than the RRE. Rev does not stimulate the translation of non-HIV RNAs and does not affect the stability or splicing of target RNAs. Using a luciferase assay reporter system, we show that Rev is also able to enhance the translation of Loop A containing receptor RNAs in COS-1 cells. The effect of Rev is most striking in cellular systems and published work indicates that Rev may influence the cellular localisation of target RNAs. Accordingly, we developed a system for the detection of Rev and genomic RNA molecules in cells. COS-1 cells were transfected with a packaging-deficient proviral plasmid. Rev protein was detected via immunofluorescence. Genomic RNA was detected using a biotinylated complementary RNA probe, which was in turn detected by a fluorophore-conjugated streptavidin molecule. This system was optimised for the detection of both targets through a number of approaches. However, the predominant nuclear localisation of Rev proved to be prohibitive for the investigation of Rev-RNA interactions in the cytoplasm. We propose that during the early phase of viral gene expression, small amounts of Rev stimulate translation but as levels increase and the focus shifts to particle production, high levels of Gag and Rev inhibit translation, contributing to Rev’s tight control of the early to late shift in viral gene expression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available