The two subtypes of the Human Immunodeficiency Virus, HIV-1 and HIV-2, differ in the way that they package their genomic RNA with the structural Gag protein. HIV-1 packages its genomic RNA predominantly in a <i>trans</i> manner, and can cross-package HIV-2 RNA in co-transfections. Conversely, HIV-2 is not capable of packaging HIV-1 RNA reciprocally, and packages its own RNA using a co-translational mechanism. Chimeric viruses where the RNA-binding domain of HIV-1 is substituted into the structural of Gag protein of HIV-2 appear to transfer the <i>trans</i> packaging phenotype, but this function is only maximally present when the adjacent p2 peptide is also included. The aim of the work presented here was to investigate possible direct and indirect effects of the p2 domain in RNA-protein capture in HIV-1 and HIV-2. Chimeric Gag proteins were expressed using bacterial systems and their interaction with <i>in vitro</i> transcribed RNA of the HIV leader sequences investigated using GST-Pulldown assays, UV-crosslinking and mobility shift assays. Evidence is presented that is consistent with a complex and possibly cooperative binding interaction occurring between Gag and the packaged RNA, which correlates to some extent with the presence of the p2 domain. The subcellular location of the Gag protein was investigated using transfection with Gag-expressing and viral constructs, following by confocal microscopy of the immunostained cells, and no difference was found that could be attributed to the p2 domain. Additionally, the replication characteristics of the chimeric viruses were examined in short and long term culture. All chimeric viruses were defective in short term culture, but one culture did produce a replication-competent strain on long-term passage. This strain had wild type replication kinetics, and sequencing of the revertant virus revealed a single-base substitution in the highly basic region of the MA domain of Gag.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596569 |
| Date | January 2004 |
| Creators | Bennett, N. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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