Rapamycin does not affect LV binding but enhances LV uptake into the cytoplasm. (A) Human cord blood CD34⁺ cells were treated with CG-UbiC-EGFP, MOI = 25, at 4°C (to prevent endocytosis and limit binding to the cell surface) for 2 hours, in the presence of 10 μg/mL rapamycin or DMSO. After transduction 60 000 to 80 000 cells were lysed and p24 protein content was determined to assess LV binding (left), and the remaining ∼10 000 cells were cultured at 37° for 10 days and evaluated for EGFP marking to determine transduction efficacy (right). Data are shown for 2 independent experiments with different cord blood donors, each done in triplicate. (B) Human cord blood CD34⁺ cells were prestimulated and treated with rapamycin or DMSO for 12 hours, and cell-surface LDL receptor (LDLR) and α-dystroglycan (α-DG) levels were determined by flow cytometry. (C) Human cord blood CD34⁺ cells were treated with DMSO (black triangles) or 10 μg/mL rapamycin (red circles) after allowing LVs to either prebind to the cell surface for 2 hours at 4°C or (D) preinternalize for 2 hours at 37°C followed by cleaving off external LVs with 0.05% trypsin (to remove bound LVs that have not internalized). Control represents same cells transduced under standard conditions outlined in Figure 1A. Data are duplicate or triplicate transductions from 1 experiment representative of 3 experiments with different cord blood donors. (E) Human cord blood CD34⁺ cells were transduced in the presence or absence of indicated concentrations of rapamycin, with CG-UbiC-mCherry, MO I = 25, carrying the BLAM-Vpr protein. After a 6-hour transduction, cells were loaded with the BLAM substrate CCF4-AM, and LV entry was quantified by flow cytometric detection of cells exhibiting cleaved CCF4. Data are pooled from 4 independent experiments, each with different cord blood donors. (F) Human cord blood CD34⁺ cells were transduced with CG-UbiC-EGFP, MOI = 25, and the following HIV-1 reverse-transcription products were quantified by qPCR: strong-stop DNA (Early RT), full-length DNA (Late RT), and 2-long terminal repeat circles. (G) Ratios between each pair of adjacent reverse-transcription products from panel F were calculated. Black, DMSO only; red, 10 μg/mL rapamycin. Data are pooled from 4 independent experiments, each with different cord blood donors. For all panels, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test (A,C,D-E) or paired Student t test (F-G). LTR, long terminal repeat.