FIG 8.

Characterization of Envs on virus particles from infected T cells. (A) HEK 293T cells were transfected with pNL4-3.env infectious molecular clones expressing the Strep-tagged AD8 Bam or E.1 Bam Envs. Addition of the Strep tag to the Env C terminus does not affect neutralization sensitivity nor Env antigenicity on virus particles (data not shown). Seventy-two hours later, the cell supernatants were clarified, and 50% infective dose (ID₅₀) values were determined using TZM-bl target cells. C8166-R5 cells were then infected with virus particles at a multiplicity of infection of 0.1. Cells were washed 5 to 16 h after infection and resuspended in fresh medium. Fresh medium was supplemented at 3 days after infection. Six to 7 days after infection, virus particles were collected, purified and Env antigenicity was analyzed as described in the Fig. 2C legend. (B) 293T cells were transfected with the pNL4-3.E.1 Bam Strep infectious molecular clone with or without a CD4-expressing plasmid at a 1:0.1 weight ratio. Seventy-two hours later, virus particles were collected, purified, and Env antigenicity was analyzed as described in the Fig. 2C legend. (C) 293T cells were transfected with pNL4-3.env IMCs expressing the unmodified AD8 Bam Env (designated –D368R) or an AD8 Bam Env with a D368R change, which decreases CD4 binding (designated +D368R) (156). Seventy-two hours later, virus particles were collected and purified; Env antigenicity was analyzed as described in the Fig. 2C legend. The results are representative of those obtained in at least two independent experiments. The means and standard deviations of the results from the Western blots shown in the left panels of A to C are reported in the bar graphs (right panels). The significance of the difference between the wild-type AD8 Bam Env and E.1 Bam Env (A), between VLP Envs made in the absence or presence of CD4 (B), or between –D368R and +D368R (C) was evaluated by a Student's t test; *, P < 0.05; ***, P < 0.001. In C, some of the P values for results that approach but do not achieve significance are shown.