Target cell-binding ability of pseudotypes bearing mutant proteins. (A and B) Cell-free NL4-3R-E-Luc reporter viruses were produced from cotransfection with pcDNA3 (marked as the control) or with each of WT and mutant E1E2 plasmids, and normalized by RT activity prior to incubation with Huh7 cells. One h post-incubation, cells were successively incubated with a rabbit anti-E2 antibody and FITC-conjugated anti-rabbit IgG, fixed with paraformaldehyde, and then analyzed by flow cytometry (A). The relative binding ability of mutant pseudotypes is expressed as a percentage of that of the WT pseudotype. Results were quantified from three individual experiments with the standard deviation shown (B). (C) Huh7 cells were incubated with medium, HIV-1 particles without E1E2 (marked as the control), and the WT pseudotype, respectively. Cells were then divided into two groups; one group of cells was separately immunostained with a rabbit anti-HIV-1 p24 or rabbit anti-E2 and fixed, and immunostained cells were analyzed by FACS (top panel). Another group of cells was fixed, permeabilized with paraformaldehyde, and immunostained with rabbit anti-p24 and anti-E2, respectively, followed by FACS analysis (bottom panel).