FIG 4.

Effects of State-1-stabilizing and -destabilizing changes on virion Env. (A) Neutralization of the AD8 Bam and AD8 Bam 197 HT N viruses by the indicated Env ligands was measured as described in the Fig. 3B legend. The 50% inhibitory concentrations of the Env ligands are reported in μg/mL except for BNM-III-170 (in μM). (B) 293T cells were transfected with pNL4-3.env IMCs expressing the AD8 Bam Env, the State-1-destabilized AD8 Bam 197 HT N Env and the State-1-stabilized Envs Tri Bam and AE.1 Bam. Seventy-two hours later, virions were purified and the antigenicity of Env on virus particles was analyzed as described in the Fig. 2C legend. (C) 293T cells were transfected with the pNL4-3.env IMCs expressing soluble versions of gp120 (sgp120) from the indicated Envs. Forty-eight hours later, the cell supernatant containing secreted gp120 was collected, filtered through a 0.45 μm membrane and incubated with the indicated antibodies and protein A-agarose beads for 2 h at room temperature. The beads were washed and Western blotted with a goat anti-gp120 antibody. (D) Purified virus particles were incubated with the crosslinker BS3 at the indicated concentrations for 30 min at room temperature, after which the reactions were quenched and samples were analyzed by reducing SDS-PAGE and Western blotted with a goat anti-gp120 antibody. The results are representative of those obtained in two independent experiments. The means and standard deviations of the results in A and B are shown in the bar graphs. The significance of the difference in antibody binding between Env mutants and AD8 Bam Env was evaluated by a Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.