Codisplay of cytoplasmic tail deletion mutants of H and F gps on HIV vectors results in high titers. 293T cells were transfected with HIV-gagpol, HIV-GFP vector, and the combination of H and F gps: Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt. RDTR was used as a control gp. (A) Images show GFP expression and syncytia formation of 293T producer cells 24 hours after transfection. Scale bar represents 1000 μm. (B) At 24 and 48 hours after transfection, cells were fixed and stained with Giemsa staining solution, and syncytia formation (percentage cell fusion) was quantified. (C) 293T cells were incubated with serial dilutions of GFP-encoding HIV vectors pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, HΔ24/Fwt, or RDTR gps. Vector supernatant was harvested at 24 or 48 hours after transfection. Cells were analyzed for GFP expression 72 hours after transduction by FACS. Titers are expressed as TU/mL (means ± SD; n = 6). (D) HIV-GFP vectors were pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt and purified over a sucrose cushion. Subsequently, viral gps, Hwt and HΔ24, were detected by Western blot with an antibody recognizing H ecto-domain; incubation with an anti-HIV p24 antibody was used to reveal HIV capsid (CA). (E) HIV-GFP vectors were pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt. A cell-binding assay was performed using the different vector preparations, concentrated by low-speed centrifugation. Both 293T cells (CD46⁺, SLAM⁻) and control CHO cells (CD46⁻, SLAM⁻) were incubated with the indicated HIV vector pseudotypes for 1 hour. After washing, bound viral particles were detected using conformational antibodies directed against the ectodomain of H gp (anti-H) or F gp (anti-F). Data shown are representative of 3 experiments.