2.10. Cell and Virus Culture Conditions.

All cell cultures were maintained at 37 °C in an atmosphere containing 5% CO₂. HEK-293T cells and TZM-bl cells^46,50,51 were grown in Dulbecco’s Minimal Essential Medium (DMEM; Sigma-Aldrich, Inc., St. Louis, MO) containing 10% fetal calf serum (FCS), 10 mM HEPES, and antibiotics (100 U of penicillin/mL, 100 μg of streptomycin/mL). PBMC were isolated from multidonor buffy coats from healthy HIV-seronegative donors, by centrifugation onto Ficoll-Hypaque, mitogen stimulated as previously described,⁵² and maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, antibiotics (100 U of penicillin/mL, 100 μg of streptomycin/ml), and 100 U of interleukin-2/mL. The laboratory-adapted isolate HIV-1 YU.2 was passaged through activated PBMCs for 11 days. Pseudovirus stocks of PVO4 and CAP210 were obtained by cotransfection of HEK-293FT cells with pSG3^Δenv and either PVO4 or CAP210 plasmid, and subsequently the culture media supernatants containing the viral particles were harvested 48 h post-transfection. The viral solutions were sterile filtered and stored in −80 °C until use.