The molecular mechanisms involved in the regulation of protease (PR) activity and human immunodeficiency virus type 1 (HIV-1) viral maturation are incompletely elucidated. To better understand the importance of the cleavage event between HIV-1 PR and reverse transcriptase (RT), we have selectively mutagenized specific residues at the junction between these genes to produce a PR-RT fusion protein in the context of a full-length proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR:RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein not only remained functionally active, bid also processed HIV-1 precursor proteins with slightly increased efficiency as shown in time-course experiments in transfected COS-7 cells. In contrast, the RT component of the fusion protein was active at wild-type levels in in vitro and endogenous RT assays. Electron microscopy revealed that mutant viruses containing the cleavage site mutation between PR and RT possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20-times less infectious than Wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N-terminus of PR, a cleavage site mutation between PR and RT did not prevent proteolytic processing and abolish infectivity; rather, viruses containing PRAT fusion proteins were viable, in agreement with the notion that C-terminal liberation of PR is not as critical for
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35840 |
| Date | January 1999 |
| Creators | Cherry, Elana. |
| Contributors | Wainberg, Mark A. (advisor) |
| 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: 001652232, proquestno: NQ50069, Theses scanned by UMI/ProQuest. |
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