Isolation of HIV-1 cores.

HIV-1 pseudovirions were used for the isolation of HIV-1 cores. Pseudovirus particles were produced by a previously described protocol (20, 21, 30). Briefly, approximately 10⁶ human embryonic kidney (HEK) 293T cells were transfected with 2.5 μg of ΔEnv IN-HIV-1 plasmid (DHIV3-GFP-D116G) (31) using 10 μg of polyethylenimine (PEI; branched; molecular weight [MW], ∼25,000; Sigma-Aldrich). After 20 h, the medium was replaced with fresh medium (Dulbecco modified Eagle medium supplemented with 10% heat-inactivated bovine serum, 1% penicillin-streptomycin, and 1% glutamine), and the cells were incubated at 37°C in 5% CO₂. After 6 h, the supernatant was harvested, centrifuged at 1,000 rpm for 10 min, and filtered through a 0.45-μm-pore-size filter. The virus-containing supernatant was concentrated by ultracentrifugation in an SW-28 rotor (25,000 rpm, 2 h, 4°C) using OptiPrep density gradient medium (Sigma-Aldrich). Part of the supernatant containing pelleted viruses was collected, mixed with 10 ml of TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA [pH 7.4]), and added to the 100-kDa molecular mass cutoff Vivaspin 20 centrifugal concentrators (100,000 MWCO; Sartorius AG, Germany). The mixture was centrifuged twice at 2,500 × g for 25 to 30 min at 4°C, until the supernatant level in the concentrators reached 200 to 300 μl.

From concentrated virus-containing supernatant, viral cores were isolated using a previously described protocol (32), with modifications. Briefly, ∼40 μl of purified HIV-1 pseudovirus particles was mixed with an equal amount of 1% Triton-X diluted in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0) and incubated for 2 min at 4°C. The mixture was centrifuged at 13,800 × g for 8 min at 4°C. After removing the supernatant, the pellet was washed twice by adding ∼80 μl of MOPS buffer and centrifuging at 13,800 × g for 8 min at 4°C. The pellet was resuspended in 10 μl of MOPS buffer and used immediately for AFM studies.