Fusogenicity of PA-R5 and A-R5 Envs cloned from the cross-sectional panel of primary R5 HIV-1 isolates. Fusion assays were performed using 293T effector cells expressing PA-R5 and A-R5 Envs shown in Fig. 1B and Cf2-Luc target cells expressing CD4 and CCR5, as described in the Methods. Cells were harvested at 2, 4, 6, 8, 10 and 12 h post-mixing and assayed for luciferase activity (A). 293T effector cells were stained for cell surface Env expression using pooled AIDS serum and analysed by flow cytometry, as described in the Methods (B). The data were stratified by different maximal levels of fusion scored as +/-, +, ++, and +++, which correspond to <10-fold (very low), 10- to 20-fold (low), 20- to 40-fold (moderate), and >40-fold (high) increases in luciferase activity above background levels, respectively (C). Box plots were constructed from mean values of duplicate experiments with each Env using Prism version 4.0c (GraphPad Software, San Diego, CA.). 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.