Functional gelsolin overexpression impairs HIV-1 entry and infection in permissive lymphocytes, regardless of viral tropism. (A) CD4 and CXCR4 (top images) or CCR5 (bottom images) distribution in uninfected CEM.NKR-CCR5 cells transfected with free EGFP (pEGFP-N1) or gelsolin-EGFP. Merged confocal microscopy images show the co-localization of CD4-CXCR4/gelsolin-EGFP or CD4-CCR5/gelsolin-EGFP at the plasma membrane. Scale bar = 5 μm; one representative experiment of three is shown, always analyzing 200 cells. (B) Flow cytometry analysis of the effect of gelsolin-EGFP or EGFP overexpression (control, 100% viral receptor expression in pEGFP-N1-transfected cells) on CD4, CXCR4 and CCR5 cell-surface expression. (C) Effect of gelsolin-EGFP overexpression on SDF-1α-induced CXCR4 endocytosis. Control (EGFP-transfected cells) and gelsolin-EGFP expressing cells were exposed to SDF-1α (50 nM and 200 nM), as described in Methods. CXCR4 expression was subsequently analyzed by flow cytometry in control/EGFP + and gelsolin-EGFP + cells using PE-conjugated specific mAbs against cell-surface CXCR4. The data are expressed as a percentage of CXCR4 expression in the absence of SDF-1α (control). (D) Luciferase assay of viral entry and infection by X4- and R5-tropic HIV-1 viral particles in gelsolin-EGFP transfected cells (EGFP-expressing cells were used as controls, and represent 100% viral entry and infection). All the data were corrected using the non-productive infection values (baseline) obtained in the presence of a neutralizing anti-CD4 mAb (5 μg/mL) under the same experimental conditions. A luciferase-based assay, with equal inputs of pseudotyped VSV-G virions was used to control the specificity of gelsolin-mediated effects on HIV-1 viral entry and infection. These data were corrected with non-productive baseline values obtained using non-infective virus-like particles lacking VSV-G-Env under the same experimental conditions. In B-D data are mean ± s.e.m. of three independent experiments carried out in triplicate: *** p < 0.001, Student’s t-test.