Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells
The activity #in vivo# of HIV-1 proteinase (PR) was analysed in the baculovirus expression system, using eight different constructs of the #prt# gene under the control of polyhedrin (PH) promoters of various strengths. None of the active PRS was expressed in substantial quantities, and only PH-fused and/or non-functional PR mutants accumulated in high amounts in insect cells. However, enough PR activity was generated from a lengthened PR construct in insect cells to process Gag polyprotein substrate co-expressed in the same cells in trans. Fusion of the first 58 residues from the PH sequence to the PR N terminus did not significantly change its activity and specificity of cleavage of the Gag substrate. When analysed under mild denaturing conditions, PH-fused or unfused fulllength PR point mutants, as well as PH-fused or unfused C-terminal deletion mutants, showed a propensity to multimerize, with a predominant occurrence of dimers. The incorporation of PR into Gag particles was studied using eight Gag-PR fusion, constructs, all containing a non-functional PR mutant. The PR domain was fused to the C-terminal p6 domain of Gag (p6Gag), or translated in frame with NCp7 (as in frameshifted Gag-Pol polyprotein) and followed by downstream sequences of increasing lengths from the Pol domain or the bacterial Bêta-galactosidase. The results suggested that the presence of the p6Gag domain was detrimental to the encapsidation of polyprotein-embedded PR.
| Main Authors: | , , , |
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| Format: | article biblioteca |
| Language: | eng |
| Subjects: | L73 - Maladies des animaux, U30 - Méthodes de recherche, maladie de l'homme, maladie immunologique, virus, Baculoviridae, activité enzymatique, protéolyse, protéine microbienne, Retroviridae, Virus de l'immunodéficience humaine, http://aims.fao.org/aos/agrovoc/c_29198, http://aims.fao.org/aos/agrovoc/c_3806, http://aims.fao.org/aos/agrovoc/c_8262, http://aims.fao.org/aos/agrovoc/c_9053, http://aims.fao.org/aos/agrovoc/c_2604, http://aims.fao.org/aos/agrovoc/c_6261, http://aims.fao.org/aos/agrovoc/c_12537, http://aims.fao.org/aos/agrovoc/c_6541, http://aims.fao.org/aos/agrovoc/c_37855, |
| Online Access: | http://agritrop.cirad.fr/401074/ http://agritrop.cirad.fr/401074/1/401074.pdf |
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