During HIV-1 assembly tRNALys isoacceptors are selectively packaged into HIV-1. One of the tRNALys isoacceptors, tRNA Lys3 serves as a primer for the reverse transcriptase-catalyzed synthesis of minus strand, strong stop cDNA. Lysyl-tRNA synthetase (LysRS) a tRNA Lys-binding protein is also selectively package into HIV-1, making it a candidate for being the signal which targets tRNALys for incorporation into viruses. We have tested whether the cytoplasmic tRNA Lys/LysRS interaction is required for selective packaging of tRNA Lys into HIV-1 by constructing anticodon mutations within the anticodon domain of tRNALys3. The anticodon is the major binding domain for tRNALys/LysRS interaction. We found that the ability of tRNALys3 to bind to LysRS, and be aminoacylated was directly correlated with its ability to be incorporated into HIV-1. / However it was not clear from these results if in addition to the tRNALys/LysRS interaction, tRNA aminoacylation was required for selective packaging of tRNA Lys3. To investigate this, we constructed and expressed two mutant LysRS species. One has lost its aminoacylation activity because of a deletion within the catalytic core. The other mutant LysRS has a reduced ability to bind to tRNALys, because it contains a deletion within tRNA Lys binding site. This mutant LysRS does not have the ability to facilitate tRNALys packaging into the virus, while the mutant LysRS which binds to tRNALys, but fails to aminoacylate it, does facilitate tRNALys packaging, indicating that tRNALys aminoacylation is not required for its packaging into viruses. / LysRS is carried into the viruses by its interaction with Gag. We have mapped the sites of interaction between HIV-1 Gag and LysRS using in vivo and in vitro techniques. We find that Gag sequences within the C-terminal domain of CA which contains the CA dimerization site, interact with LysRS sequences which include motif 1, which contains the LysRS dimerization site.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84263 |
| Date | January 2003 |
| Creators | Javanbakht Rezai, Mohammad Hassan |
| 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: 002032099, proquestno: AAINQ98279, Theses scanned by UMI/ProQuest. |
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