The Gag protein of human immunodeficiency virus type 1 (HIV-1) contains a 14 amino acid region termed SP1 that is located between the capsid (CA) and downstream nucleocapsid (NC) sequences, and plays an active role in Gag assembly. This activity of SP1 involves its amino-terminal residues that, together with adjacent CA residues, constitute a putative (x-helical structure spanning Gag residues from positions 359 to 371. However, the underlying mechanisms are mostly unclear. The major goal of this PhD study was to understand the role of SP1 in HIV-1 Gag multimerization, membrane binding and viral RNA packaging. In addition to the reported function of the SP1 region in virus assembly, we further mapped the assembly determinants within SP1 to H359, K360, L364, A367, and M368. Detailed analysis of the L364A and M368A mutations revealed that they not only affected Gag multimerization, but also Gag-membrane association. We propose that the latter defect is a result of the former. Interestedly, the membrane binding defect associated with M368A can be corrected either by changing NC to the leucine zipper (LZ) motif derived from the yeast transcription factor GCN4 or by a L364A second-site mutation, although neither of them restored wild-type levels of particle production to the M368A Gag. These results suggest that SP1 may affect events of virus assembly subsequent to Gag-membrane binding such as capsid morphogenesis or virus budding. We also studied the R362A mutation that is located within a short 359-HKAR-362 basic fragment. Results of RNase protection assay, native Northern blots as well as filter-binding assays showing reduced packaging of viral RNA into the R362A mutated viruses. These data reveal the function of the C-terminal disordered sequence of CA in HIV-1 RNA packaging. Further analysis of the BIV CA/NC spacer region demonstrated the key role of this region in BIV Gag assembly. In summary, our results demonstrate that the SP1 sequence regulates Gag multimerization, Gag-membrane binding as well as viral RNA packaging.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111821 |
| Date | January 2005 |
| Creators | Guo, Xiaofeng, 1976- |
| 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 (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002326128, proquestno: AAINR25162, Theses scanned by UMI/ProQuest. |
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