Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells
| Autor: | M Royer, J Tournier, B Gay, P Boulanger, M Bardy |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Baculoviridae
viruses Mutant L73 - Maladies des animaux Protéine microbienne Substrate Specificity law.invention HIV Protease law Polyhedrin Promoter Regions Genetic Virus de l'immunodéficience humaine biology Virus Recombinant DNA Electrophoresis Polyacrylamide Gel Activité enzymatique Recombinant Fusion Proteins Gene Products gag Spodoptera Transfection Cleavage (embryo) Cell Line Protéolyse Viral Proteins Maladie de l'homme Virology Animals Humans Gene Viral Structural Proteins Maladie immunologique Promoter biology.organism_classification Occlusion Body Matrix Proteins Molecular biology N-terminus Kinetics Retroviridae HIV-1 U30 - Méthodes de recherche |
| Zdroj: | Journal of General Virology |
| ISSN: | 1465-2099 0022-1317 |
| DOI: | 10.1099/0022-1317-78-1-131 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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