FIG 5.

Shedding of gp120 from Env on virus particles. (A) 293T cells were transfected with the pNL4-3.AD8 Bam plasmid. Forty-eight to seventy-two hours later, the cell supernatants were collected, filtered through a 0.45-μm membrane and centrifuged at 100,000 × g for 1 h at 4°C. Virus pellets were resuspended and incubated with four-domain soluble CD4 (sCD4) or the CD4-mimetic compound BNM-III-170 at the indicated concentrations and temperatures for 1 h. Virus particles were again pelleted and the supernatants containing shed gp120 were incubated with GNL beads for 2 h at room temperature. Beads were washed and Western blotted with a goat anti-gp120 antibody. The percentage of the gp120 Env on the input virus that was detected in the supernatants is plotted in the graphs on the right, which are derived from a representative experiment. (B) Shedding of gp120 from different Envs after a 1-h room temperature incubation with the indicated concentrations of BNM-III-170 was analyzed as described in A. (C) Purified virus particles were incubated at different temperatures for different lengths of time. Shed gp120 was then analyzed as described in A. (D) Purified virus particles with the AD8 Bam Env were incubated on ice in the presence of 10 μM BMS-806 or the equivalent concentration of the DMSO solvent for different lengths of time. Shed gp120 was then analyzed as described in A. (E) Purified virus particles containing different AD8 Env variants were incubated on ice for the indicated lengths of time and shed gp120 was then analyzed as described in A. Except for A, the results shown are representative of those obtained in two independent experiments. In B-D, the means and standard deviations are reported. The significance of the difference between DMSO- and BMS-806-treated samples (C) or between Tri Bam and AE.1 Bam Envs compared to AD8 Bam Env (D) was evaluated by a Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.