Figure 6
IMB-LA inhibits HIV-1 infection in BST-2-expressing cells.
(A) 293 T cells and 293T-BST-2 were co-transfected with 300 ng pNL4-3luc(R-E-), 210 ng pHIT/G and 200 ng pcDNA3.1-BST-2-HA. The Vpu-negative pNL4-3luc (−Vpu) was used as a control. 24 h post-transfection, cells were treated with DMSO or 5 μM IMB-LA for 24 h. Cellular content of Gag and BST-2-HA were analyzed by Western blot using anti-p24 antibody and anti-HA antibody, respectively. β-actin was detected as a control (left panel). Amounts of viruses produced from the transfected cells were determined by p24CA ELISA (middle panel). The progeny viruses containing the same amount of p24 were used to infect SupT1 cells and HIV-1 infection was determined by measuring firefly luciferase activities (right panel). (B) HeLa and HeLa-BST-2-KD cells were transfected with 500 ng pNL4-3luc(R-E-) or pNL4-3luc (-Vpu) and 350ng pHIT/G. Protein expression in cells (left panel), amounts of viruses (middle panel) and infectivity of the progeny viruses (right panel) were determined as described in Fig. 6A. (C) 5 × 105 HeLa or HeLa-BST2-KD cells were transfected with 0.3μg NL4-3deltaLuc and 0.3 μg Env and then were treated with DMSO or IMB-LA, respectively. The supernatant was used to infect TZM-bl cells and luciferase activity was measured. (D) TZM-bl cells were infected with wild type NL4-3 viruses and then incubated with the indicated compounds, followed by luciferase activity measurement. (E) 293T cells were transfected with pcDNA3.1-BST-2 and either HIV-1(WT) or HIV-1(delVpu), followed by treatment with either DMSO or 5 μM IMB-LA. Released particles were harvested from culture supernatants before (top panel) and after (bottom panel) subtilisin strip, respectively and then subjected to Western blot analysis. (F) HEK293T cells were co-transfected with pcDNA3.1-BST-2-HA, pNL4-3luc(R-E-) and pHIT/G as described in Figure 6 (A). Cells and virions were subjected to western blot analysis. (anti-p24 and anti-HA, as indicated). (G) The HeLa-BST-2-KD cells were exposed to virion supernatants containing the same amount of p24. 24 h later, cells were treated with IMB-LA for 24 h. Firefly luciferase activities in cell lysate were determined. Each bar represents the mean and standard deviation of at least three independent experiments. **denotes P < 0.01. Full-length blots/gels are presented in Supplementary data.
