Preparation of VLP-Containing HXB2 env

VLP were propagated using human embryonic kidney, 293T cells (ATCC). Cells were maintained in Dulbecco’s Modified Eagle medium with 10% Fetal Bovine Serum. VLP-containing HXB2 envelope was produced by transient transfection of HEK 293T cells with pGag-EGFP and pHXB2 env using Lipofectamine 2000 transfection reagent (Life Technologies). Also, 10⁷ cells in T175 flasks were transfected with 30 µg HXB2 envelope, 60 µg pGag-EGFP, and 360 µL Lipofectamine 2000 transfection reagent in serum/antibiotic-free medium. After 3–4 h of incubation at 37°C, the culture medium was replaced with DMEM + 10% FBS. VLP-containing supernatant was collected 72 h after transfection and clarified by centrifugation at 2,000 × g for 10 min. Clarified supernatant was further purified of cellular debris by 0.45 µm filtration. Purification of assembled VLP was completed by ultracentrifugation through a 20% sucrose pad at 122,000 × g for 2 h at 4°C. The VLP pellet was resuspended in filtered PBS.