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Journal of Virology, August 2000, p. 7238-7249, Vol. 74, No. 16
Departments of Pediatrics and Microbiology and Immunology,
Vanderbilt University School of Medicine, Nashville, Tennessee
Received 18 January 2000/Accepted 22 May 2000
Human immunodeficiency virus (HIV) type 1 particles assemble at the
plasma membrane of cells in a manner similar to that of the type C
oncoretroviruses. The Pr55Gag molecule directs the assembly
process and is sufficient for particle assembly in the absence of all
other viral gene products. The I domain is an assembly domain that has
been previously localized to the nucleocapsid (NC) region of Gag. In
this study we utilized a series of Gag-green fluorescent protein (GFP)
fusion proteins to precisely identify sequences that constitute the
N-terminal I domain of Pr55Gag. The minimal sequence
required for the I domain was localized to the extreme N terminus of
NC. Two basic residues (arginine 380 and arginine 384) within the
initial seven residues of NC were found to be critical for the function
of the N-terminal I domain. The presence of positive charge alone in
these two positions, however, was not sufficient to mediate the
formation of dense Gag particles. The I domain was required for the
formation of detergent-resistant complexes of Gag protein, and confocal
microscopy demonstrated that the I domain was also required for the
formation of punctate foci of Gag proteins at the plasma membrane.
Electron microscopic analysis of cells expressing Gag-GFP fusion
constructs with an intact I domain revealed numerous retrovirus-like
particles (RVLPs) budding from the plasma membrane, while I
domain-deficient constructs failed to generate visible RVLPs. These
results provide evidence that Gag-Gag interactions mediated by the I
domain play a central role in the assembly of HIV particles.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mapping and Characterization of the N-Terminal I
Domain of Human Immunodeficiency Virus Type 1 Pr55Gag
*
Corresponding author. Mailing address: Pediatric
Infectious Diseases, Vanderbilt University, D-7235 MCN, Nashville, TN
37232-2581. Phone: (615) 322-2250. Fax: (615) 343-9723. E-mail:
paul.spearman{at}mcmail.vanderbilt.edu.
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