FIG 4.
Mechanisms of HIV-1AD8 escape from BNM-III-170 and sCD4-Ig. (A) BNM-III-170-induced shedding of gp120 from virions with the indicated Envs was measured. Virions produced transiently from HEK 293T cells transfected with pNL4-3-AD8 proviral constructs were harvested, clarified by low-speed centrifugation, filtered (0.45-μm) and pelleted at 14,000 × g for 1.5 h at 4°C. The virus pellet was resuspended in 1× PBS and incubated with the indicated concentrations of BNM-III-170 for 2.5 h at 37°C. The viruses were then pelleted; the virus pellet was lysed and the supernatant containing shed gp120 was incubated with Galanthus nivalis lectin (GNL) beads. The viral lysates and the protein captured on the GNL beads were Western blotted. (B) gp120 shedding in the presence of BNM-III-170 was measured by quantifying the gp120 bands in the Western blots shown in A. The shed gp120 was calculated according to the formula: gp120 supernatant/(gp120 supernatant + gp120 virions). (C) The correlation between BNM-III-170 inhibition of pseudovirus infection and BNM-III-170-induced gp120 shedding is shown. The amount of gp120 shedding at a BNM-III-170 concentration of 50 μM, measured as in panel B, was used in the correlation analysis. The Spearman rank-order correlation coefficient (rS) and P value are shown. (D) Precipitation of virion Env by sCD4-Ig was evaluated as an indication of the affinity of sCD4-Ig for the Env variants. Virion pellets prepared as in panel A were resuspended in buffer containing the indicated concentrations of sCD4-Ig and incubated for 1.5 h at room temperature. After pelleting, the viruses were washed, lysed and incubated with protein A-agarose beads. The proteins captured on the beads were Western blotted. The results shown are typical of those obtained in at least two independent experiments. (E) The ratio of the virion gp120 glycoproteins precipitated by sCD4-Ig to the input gp120 glycoproteins was calculated from the experiment shown in panel D. The ratio is shown as a function of the sCD4-Ig concentration used for the immunoprecipitation assay.