Fig. 6. Sensitivity of the heavily mutated Env variants to ligand binding and neutralization.

Sensitivity of NL4-3 7XEnv to (A) NAbs recognizing the CD4-bound conformation and (B) sCD4. TZM-bl cells were exposed to 100 TCID₅₀ of viruses in the presence of various concentrations of NAbs or sCD4. Luciferase activity was measured at 48 hours after infection. (C to E) NAb/CD4-Ig binding to Env on the cell surface. 293T cells transfected with the indicated WT or 7XEnv pBR43IeG clones were preincubated with (C) CD4-Ig, (D) VRC03, and (E) 17b at 37°C for 30 min. The cells were washed, and Alexa Fluor 647–conjugated anti-human IgG was used to detect bound antibodies. For 17b binding, Env-expressing cells were treated with the indicated concentrations of sCD4. Alexa Fluor 647 signals were normalized by eGFP signals to calculate the Ab binding efficiency. (F) Effect of the 7XEnv mutations on cold sensitivity. RT-normalized viruses were incubated at 4°C for the indicated times and frozen at −80°C. The viral aliquots were quickly thawed, and infectivity was measured using TZM-bl cells. (G) CD4-Ig binding to CH185 Env on the cell surface. 293T cells transfected with the indicated Env mutants were preincubated with CD4-Ig at 37°C for 30 min. The cells were washed, and Alexa Fluor 647–conjugated anti-human IgG was used to detect bound antibodies. To detect p24 in the cells, the transfected cells were fixed and permeabilized and then stained with FITC-conjugated anti-p24 Ab. (H) Cold sensitivity of the CH185 Env mutants. RT-normalized viruses were incubated at 4°C for the indicated times and frozen at −80°C. The viral aliquots were quickly thawed, and infectivity was measured using TZM-bl cells. Data from at least three independent experiments are shown as means ± SEM. *P < 0.05, unpaired t test.