Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates
Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escheri...
Saved in:
Published in | The Journal of biological chemistry Vol. 279; no. 30; pp. 31964 - 31972 |
---|---|
Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
23.07.2004
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation
of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism
of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 °C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the
virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer
characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles
in the E. coli cells. In contrast, expression at 30 °C resulted in the production of only the 60 S form of Gag multimer, and crescentshaped
structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 °C, also produced the high order of Gag multimers with
some 60 S multimers, whereas the 30 °C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked
so as not to allow conformational changes, in vitro assembly reactions at 37 °C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the
E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins,
induced at 37 °C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through
the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes
in Gag. |
---|---|
Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M313432200 |