Figure 3

Binding of CAP treated and untreated HIV-1 to distinct ligands. The binding of untreated and CAP treated HIV-1 IIIB to wells coated with sCD4 or with distinct mAbs and of HIV-1 BaL to anti-V3 BaL was measured as described for Fig. 2. The % of binding corresponding to CAP treated virus was calculated based on the formula (% residual binding = [absorbance corresponding to bound CAP treated virus ÷ absorbance corresponding to bound untreated HIV-1] × 100. Absorbance corresponding to p24 antigen (5-fold dilution of the sample) from untreated HIV-1 bound to the respective ligands was in the range of 0.56 to 1.54. Absorbance corresponding to virus captured onto wells coated with control IgG was 0.046. All experiments were done in triplicate. To measure virus binding to the coreceptor CXCR4, treated and control HIV-1 IIIB recovered after PEG precipitation was mixed with 10 μg of sCD4. After 5 min at 20°C, the respective samples were divided into 2 aliquots, each of which was added to 5 × 10⁵ GHOST CXCR4 cells suspended in 100 μl PBS containing 100 μg/ml of BSA. Similar experiments were carried out with purified HIV-1 BaL, except that GHOST CCR5 cells were used. After 1 h at 4°C, the cells were pelletted and washed with ice cold PBS containing 100 μg/ml BSA. The pelletted cells were lysed for 30 min at 37°C in PBS with 1% NP40. Serial 5-fold dilutions in PBS (1:5 to 1:4.9 × 10⁷) were tested by ELISA for the p24 antigen. Changes in HIV-1 binding were determined using calibration curves relating absorbance to virus dilutions.