H/F-LVs exclusively use the CD46 receptor for high-level transduction of stimulated and quiescent hCD34⁺cells. (A) Surface staining for the measles receptors (SLAM, CD46, and nectin-4) on resting or cytokine (TPO + SCF + Flt3-L) prestimulated hCD34⁺ cells. (B) Quantification of CD46 surface expression mean fluorescence intensity (MFI) of hCD34⁺ cells at different time points of cytokine stimulation (TPO + SCF + Flt3-L). (C) Prestimulated and resting CD34⁺ cells were stained for CD46, and each was sorted for low and high CD46 expression. Obtained subpopulations were transduced with H/F-LVs in the presence of retronectin, and transduction efficiency was analyzed by FACS 3 days later. (D) Prestimulated and (E) resting hCD34⁺ cells were transduced with H/F-LVs (MOI, 10) in the absence or presence of anti-CD46, anti-SLAM, or anti-nectin-4 blocking antibodies (Ab’s). Three days after transduction, GFP expression in these cells was determined by FACS. Transduction levels are presented as GFP expression relative to the H/F-LV transduction in the absence of antibody, set to 100% (mean ± SD; n = 3). (F) Schematic representation of the different MV hemagglutinin gp’s (Hgp’s). (H) The MV vaccinal Edmonston strain Hgp contains binding residues for both SLAM and CD46. CD46-tropic mutant Hgp was engineered by mutating 1 residue of Edmonston Hgp responsible for binding/fusion through SLAM (R533A; H533). SLAM-tropic Hgp was derived from the clinical strain D4 (H-D4). H-D4-YG was obtained by introducing CD46 binding residues (481Y and 492G) into the H-D4 envelope. Prestimulated hCD34⁺ cells were transduced with H/F-LVs, H-D4/F-LVs, H533/F-LVs, or H-D4-YG/F-LVs, and GFP expression was analyzed by FACS 6 days later (mean ± SD; n = 4). ns, not significantly different for H533 and HD4-YG compared with H. Statistical analysis was performed by using paired Student t test.