U601188.pdf (11.47 MB)
Recognition of HIV-1 genomic RNA by gag
thesisposted on 2014-12-15, 10:33 authored by Ben. Maddison
In the HIV-1 infected cell, a specific recognition event has to take place between the structural polyprotein precursor known as gag, and the virion genomic RNA. This interaction is essential for ensuring that the assembling virus packages a genome. The interaction is specific as cellular RNAs and spliced genomic RNAs are largely excluded from the virus particle. Several published reports have suggested that the p55 gag protein specifically interacts with RNA sequences in the 5' untranslated region of the virion genome. This thesis describes in-vitro investigations designed to further understand this interaction with the aim of determining a minimal binding site for the HIV-1 gag protein on the genomic RNA. Using a deletion type approach the interaction was analysed by filter binding techniques with a GST-p55 gag fusion protein and in-vitro transcribed RNA. This interaction was then subsequently investigated by ribonuclease footprinting. These methodologies identified regions of RNA rich in secondary structure, between the 5' splice donor and up to and including the gag start codon, which interact with GST-p55 in solution. The location of the gag-RNA interaction in infected cells had not been established. I investigated this using subcellular fractionation techniques, UV photo affinity cross-linking of RNA-protein interactions, immunoprecipitation and RT-PCR. These experiments show that gag-RNA interactions do not occur in the nuclei of the infected cell. This interaction probably occurs at some location in the soluble cytoplasm, as gag-RNA complexes could be identified in the soluble cytoplasmic fractions after fractionation on sucrose gradients which separate out cytosol and membrane fractions. Preliminary experiments suggest that gag interacts with HIV-1 genomic RNA sequences in the cytoplasm and not subgenomic HIV-1 RNAs, as required for virion morphogenesis.
Date of award1999-01-01
Awarding institutionUniversity of Leicester