Different BST-2 mutants show different inhibitory effects on Vpu-defective or wild-type virus particle release.

(A and B) BST-2 WT NHA (12.5 ng or 25 ng), BST-2 WT IHA (12.5 ng or 25 ng), BST-2 WT NT (12.5 ng or 25 ng), BST-2 ΔN20 NHA (12.5 ng or 25 ng), BST-2 ΔN20 IHA (12.5 ng or 25 ng), BST-2 ΔN20 NT (12.5 ng or 25 ng) or the VR1012 control vector (adjusted to 25 ng) was co-transfected with 1 µg of the proviral plasmid pNL4-3 ΔVpu or pNL4-3 WT in 293T cells. At 48 h post-transfection, cultured supernatants were ultracentrifuged to concentrate the virus particles. Virions and cell lysates were analyzed by Western blotting using an anti-p24 antibody to detect viral p24 and intracellular Pr55Gag proteins, an anti-BST-2 pAb to detect BST-2 and an antibody against tubulin to assess sample loading. The blots of released p24CA were quantified using Bandscan software and normalized by tubulin levels. (C) The titration of the released infectious viruses in A and B are shown in columns below the Western blotting, which are representative of three independent experiments.