FIG. 3.

Infectivity as a function of time of addition of fusion inhibitors in a synchronized HXB2-Env pseudotyped viral infectivity assay at 37°C. (A) Fully inhibitory concentrations of sCD4 (50 μg/ml), C34 (200 nM), and N36^Mut(e,g) (50 μM) peptides and 2G12, 2F5, 4E10, and bF-3674 neutralizing MAbs (50 to 200 μg/ml) were added at the indicated time points. The experimental data (averages from four to eight experiments, with error bars representing standard deviations) were fit to a sigmoidal curve (see Materials and Methods). At 37°C, the t_1/2s of the inhibitor-sensitive state are as follows: sCD4, ≤1 min; 2G12, ≤ 1 min; 2F5, 15.0 ± 1.8; 4E10, 15.9 ± 1.7 min; C34, 19.0 ± 2.3 min; N36^Mut(e,g), 20.6 ± 2.3 min; and bF3674, 21.5 ± 2.0 min. The so-called zero time point represents data acquired by adding cold medium containing a high concentration of inhibitor to the cold cells prior to initiating fusion by raising the temperature to 37°C. The small degree of inhibition of infection observed at the zero time point for both sCD4 and 2G12 indicates that although the pseudovirus is bound to CD4-bearing cells under these conditions, the conformational change in gp120 has not yet fully taken place in the cold and some competition with sCD4 and 2G12 is still possible. (B) Comparison of infectivity as a function of time of addition of sCD4 at 30°C and 37°C. The data for sCD4 at the two temperatures are similar.