Generation of cell lines

293T and Cf2Th cells were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium with 10% tetracycline-free fetal bovine serum (DMEM-10, Clontech). Cf2Th cell lines stably expressing human CD4 and CCR5 (Cf2Th-CD4-R5) were grown in DMEM-10 in the presence of 400 μg/mL G418 and 200 μg/mL hygromycin (Invitrogen).

Cf2Th cell lines with regulatable CCR5 expression were generated in the following manner: Cf2Th cells were transfected with Tet-On® Advanced Vector (Clontech), passaged in DMEM-10 and 0.6 ug/mL puromycin, and single cells selected by limiting dilution in 96-well plates. Single-cell clones were expanded and tested for expression of the tTA protein after treatment with 0–1 ug/mL doxycyline by Western blotting of whole cell lysates with a monoclonal antibody against TetR (Clontech). tTA-expressing cells were transfected with pGL422-tetO-CCR5, passaged in the presence of 0.6 μg/mL G418, and selected for single cells. Cells were expanded and tested for doxycycline-regulated CCR5 expression via flow cytometry as follows: after incubation with R-Phycoerythrin-conjugated mouse anti-human CCR5 (PE-anti-CD195, BD Biosciences), ~1 × 10⁶ cells were analyzed with a BD FACSCanto II flow cytometer (BD Biosciences). Clones with the least background and greatest dynamic range of induced CCR5 expression were then transfected with the pcDNA-CD4 plasmid expressing human CD4, passaged in the presence of 0.025 μg/mL zeocin, and cloned. Cells were tested for doxycycline regulation of CCR5 expression and stable expression of CD4 using flow cytometry with PE-anti-CD195 and fluorescein isothiocyanate-conjugated mouse anti-human CD4, clone RPA-T4 (FITC-anti-CD4, BD Biosciences), respectively.

Two clones that express high levels of CD4 and low or high ranges of cell-surface CCR5 expression upon doxycycline treatment, herein called R5-Low and R5-High, were selected for HIV-1 adaptation to low levels of CCR5 (Fig. 1A). Levels of CCR5 were measured by QuantiBritePE (Fig. 1B, BD Biosciences) and vary within a physiological range (Fig. 1C).