Sensitivity of PA-R5 and A-R5 Envs to inhibition by T-20. Fusion assays were performed using 293T effector cells expressing PA-R5 and A-R5 Envs from the cross sectional panel of primary R5 HIV-1 isolates shown in Fig. 1A, and Cf2-Luc target cells expressing CD4 and CCR5 in the presence of 10-fold increasing concentrations of T-20 ranging from 0.001 to 10 μg per ml, as described in the Methods. IC50 (A) and IC80 (B) values were calculated by least squares analysis of inhibition curves. IC80 values were calculated instead of IC90 values, because 90% inhibition of fusion was not reached when these concentrations of T-20 were tested against some of the A-R5 Envs (data not shown). 293T effector cells were stained for cell surface Env expression using pooled AIDS serum and analysed by flow cytometry, as described in the Methods (C). Box plots were constructed from mean values of duplicate experiments with each Env using Prism version 4.0c (GraphPad Software). Boxes represent upper and lower quartiles and median scores, and whiskers represent minimum and maximum values. The data shown are representative of 3 independent experiments. P values were calculated using a nonparametric Mann-Whitney U test, and values < 0.05 were considered statistically significant.