During HIV-1 assembly, the Gag structural protein specifically encapsidates two copies of viral genomic RNA in the form of a dimer. An RNA stem-loop structure (SL1) in the 5' untranslated region, known as the dimerization initiation site (DIS), is important for dimerization and packaging of HIV-1 genomic RNA; however, the mechanisms involved are not fully understood. The major goal of this PhD study was to further understand HIV-1 RNA dimerization, and to study the role of the Gag protein in the dimerization and packaging processes. Despite the known involvement of the DIS in RNA dimerization, DIS-mutated viruses still contain significant levels of dimerized RNA, and electron microscopy studies suggest that the RNA molecules are linked at the extreme 5' end. We show here that RNA sequences on both sides of the DIS are also required for HIVA genome dimerization, suggesting that multiple RNA elements are involved. We have also examined the contribution of specific amino acids within Gag to the dimerization and packaging processes. Previous work showed that partial deletion of the DIS impacted on viral replication capacity, but could largely be corrected by compensatory point mutations within Gag. To further elucidate the mechanism(s) of these compensatory mutations, we generated DIS mutants lacking the entire SL1, or only the SL1 loop sequences, and combined these deletions with various combinations of compensatory mutations. Analysis of virion-derived RNA showed that the relevant mutant viruses contained increased levels of spliced viral RNA compared to wild type, indicating that a defect in genome packaging specificity was present. However, this defect was corrected by our compensatory mutations, and a T121 substitution in p2 was shown to be solely responsible for this activity. These results suggest that the p2 spacer peptide plays a critical role in the specific packaging of viral genomic RNA. In summary, these findings provide new insig
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85092 |
| Date | January 2004 |
| Creators | Russell, Rodney S. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002166382, proquestno: AAINR06336, Theses scanned by UMI/ProQuest. |
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