FIG. 2.
The L20K mutation within MA restores wild-type (WT) membrane binding to the L364A- and M368A-containing Gag proteins. (A) Illustration of the L20K, L20K-L364A, and L20K-M368A Gag proteins. (B) The L20K-L364A and L20K-M368A Gag proteins bind cellular membranes as efficiently as wild-type Gag. Levels of membrane-associated Gag proteins were determined by performing membrane flotationcentrifugation. Relative amounts of Gag within each fraction are summarized in the graph. (C) Subcellular distribution of Gag mutants L20K-L364A and L20K-M368A. GFP was linked to the C terminus of Gag protein, and the location of fusion proteins was determined by confocal microscopy. The images shown represent the most prevalent phenotypes. (D) Virus production in transfection experiments performed with DNA constructs that yield wild-type and mutated Gag proteins. Levels of cell- and virion-associated Gag proteins were assessed by Western blot using anti-p24 (capsid) antibodies. The HIV-1 DNA constructs tested contain a protease-negative D25A mutation and thus only produce the Pr55Gag protein when transfected into COS-7 cells.