Fig. 5. Effect of moesin-GFP fusions on HIV-1 infection and HIV-1 Env-mediated cell-to-cell fusion. (A) Flow cytometry histograms show fusion (F) after 12 hours co-culture of Env+Jurkat-Hxbc2 cells and CEM cells overexpressing GFP (control) or FL-, N- or C-moesin-GFP fusions. A representative experiment of six is shown. (B) Quantification of six independent cell-to-cell fusion experiments as shown in A; bars show means ± s.e.m. ^*P<0.01 using the Mann-Whitney U test. (C) A series of X-Gal staining showing Env-mediated membrane fusion of HeLa P4 cells, 24 hours after their lipotransfection with moesin-GFP constructs, and X4-tropic Env-HeLa 243 cells. A representative experiment of four is shown. GFP indicates transfection control for Env-induced cell fusion (100%). Quantification of three independent experiments (mean ± s.e.m., n=3) is shown in parentheses. (D) Effect of silencing endogenous ezrin and/or moesin on HIV-1 Env-mediated CEM-Hxbc2 cell fusion. When indicated, cell-to-cell fusion was inhibited by a neutralizing anti-CD4 mAb. Values are means ± s.e.m., n=4. Oligos are as indicated in Fig. 3; scrambled, non-specific RNA control sequence. (E) Confocal microscopy and quantification of Hxbc2-Env-mediated membrane fusion after 30 minutes. Localization of F-actin and endogenous ERM, and CMAC diffusion from Jurkat-derived Hxbc2 cells (blue cell to right) to the target CEM T cell (left); FL-moesin-GFP concentrates at cell-cell contacts where there is a strong accumulation of F-actin and CMAC diffuses better; N-moesin does not localize to cell contacts, impairing the redistribution of F-actin and cell-cell diffusion of CMAC; C-moesin-GFP concentrates at cell-cell contacts along with F-actin and CMAC cell-diffusion was observed. Histograms show quantification of CMAC fluorescence along the white lines (see merged pictures); segments between points 1 and 2 correspond to the cytoplasm of the target cell. The percentages represent the number of contacting cells showing redistribution per 200 cell-cell contacts counted.