FIG 3.

Effects of cytoplasmic tail clipping on Env conformation. (A) 293T cells were transfected with the pNL4-3.AD8 or pNL4-3.AD8 Bam IMCs, the latter encoding the AD8 Env with Bam (S752F I756F) changes in the cytoplasmic tail. Forty-eight to seventy-two hours later, the cell lysates and virus particles were prepared and Western blotted as described in the Fig. 1A legend. Clipping of the gp41 subunit in the virus particles was reduced by the Bam changes. (B) Seventy-two hours after transfection of 293T cells with IMCs, the cell supernatants were collected, clarified with a soft spin to remove cell debris, and used to measure the sensitivity of the viruses to inhibition. The virus-containing supernatants were incubated for 1 h at 37°C with a panel of bNAbs and pNAbs, soluble CD4-Ig or the CD4-mimetic compound BNM-III-170. The mixture was added to TZM-bl cells for 48 h, after which cells were lysed and the luciferase activity measured. The 50% inhibitory concentrations of the Env ligands are reported in μg/mL except for BNM-III-170 (in μM). (C) Antigenicity of the VLP AD8 Env with and without the Bam changes was analyzed as described in the Fig. 2C legend. The results are representative of those obtained in two independent experiments. The means and standard deviations of the results in B and C are reported in the bar graphs. The significance of the difference in antibody binding to the AD8 and AD8 Bam Envs in C was evaluated by a Student's t test; *, P < 0.05.