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J Biol Chem, Vol. 274, Issue 39, 27997-28002, September 24, 1999
From 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 °C in low salt at neutral to alkaline pH.
However, higher order of assembly required incubation at 37 °C and
was facilitated by the addition of Mg2+. 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.
In Vitro Assembly of Human Immunodeficiency Virus
Type 1 Gag Protein
, The Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108-8642, Japan and ¶ Department of
Microbiology, Osaka Medical College, Daigaku-cho 2-7, Takatsuki,
Osaka 569-8686, Japan
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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