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In vitro assembly of human immunodeficiency virus type 1 Gag protein.
In vitro assembly of human immunodeficiency virus type 1 Gag protein.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1999 Sep 24; Vol. 274 (39), pp. 27997-8002. - Publication Year :
- 1999
-
Abstract
- Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees C and was facilitated by the addition of Mg(2+). Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.
- Subjects :
- Animals
Cell Line
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Gene Products, gag genetics
Gene Products, gag ultrastructure
HIV-1 genetics
Humans
Microscopy, Electron
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
Recombinant Proteins ultrastructure
Spodoptera
Transfection
Gene Products, gag metabolism
Genes, gag
HIV-1 metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 274
- Issue :
- 39
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10488150
- Full Text :
- https://doi.org/10.1074/jbc.274.39.27997