Fig. 6. Moesin drives HIV-1 Env-mediated CD4-CXCR4 interaction and polarized redistribution, during early viral infection. (A) Effect of FL- and N-moesin constructs on rs-gp120_IIIB (10 µg/mL)-induced CD4/CXCR4 direct association. Immunoprecipitation assays were performed with anti-CD4 OKT4 mAb. Inducible co-immunoprecipitation of both receptors was measured as the ratio between the intensities of CXCR4 and CD4 immunoblotted bands. A representative experiment of three is shown. (B) Effect of the silencing of endogenous moesin on HIV-1 Env-induced direct CD4-CXCR4 interaction, in CEM cells. One representative experiment of three is shown. (C) Confocal microscopy of CD4 (anti-CD4 mAb HP2/6) and CXCR4 (biotin-conjugated mAb anti-CXCR4 12G5) in non-infected (Untreated) or 1 hour HIV-1-infected CEM cells (MOI, 1). The yz and xz planes are shown for each xy mid-section presented (arrows). Scale bars: 10 µm. (D) Percentage of colocalization of ERM and CD4, Actin or CXCR4, and of CD4 and CXCR4 in the presence of HIV-1 (MOI, 3) during a tim-course assay. (E) CD4 and CXCR4 redistribution in 1 hour HIV-1-infected cells overexpressing the different moesin-GFP proteins. xy maximal projections (left) and xy mid-sections (right) are shown. Scale bars: 10 µm. (F) Quantification of HIV-1-induced CD4-CXCR4 co-distribution in CEM T cells overexpressing FL-moesin-GFP and N-moesin-GFP constructs. (G) Quantification of HIV-1-induced CD4-CXCR4 capping in untransfected cells (Control), cells overexpressing the scrambled siRNA or lacking endogenous moesin (siRNA-moesin oligo 2). In F and G, quantification of HIV-1-induced CD4-CXCR4 capping is indicated as a percentage of each 200 and 100 cells counted, respectively, under each experimental condition.