Figure 1

Sequential analysis on the level of endogenous hBST-2 on the surface of HIV-1-infected human T cells. (A and B) MT-4 cells were stained with a mouse anti-hBST-2 antibody, and the surface expression of endogenous hBST-2 (filled in gray) was analyzed by flow cytometry as described in the Materials and Methods. Isotype IgG was used as a negative control (broken line). A representative result (A) and summarized graph (B) are shown. The level of endogenous hBST-2 on the surface of MT-4 cells (opened bar and circle) is compared to that of HeLa and HEK293 cells (filled bars and circles). MFI is represented in bars (Y-axis on left), and the percentage of hBST-2-positive cells is represented in circles (Y-axis on right, log scale). (C) The level of endogenous hBST-2 expression in HeLa, HEK293, and MT-4 cells was analyzed by Western blotting (top panel). For clear detection of hBST-2, the cell lysates were treated with glycopeptidase as described in the Materials and Methods, and the level of deglycosylated hBST-2 was analysed by Western blotting (bottom panel). The input was standardized to Tubulin, and representative results are shown. kDa, kilodalton. (D-H) MT-4 cells were infected with either wild-type or Vpu-deficient HIV-1 (MOI 0.1). Endogenous hBST-2 on the cell surface and intracellular expression of p24 were sequentially analyzed by flow cytometry, and representative profiles are shown (D). The number in the corner of the plot indicates MFI of hBST-2 on the surface of whole cells, and that in the square in the plot indicates MFI of hBST-2 on the surface of p24-postive cells. The amount of p24 in the culture supernatant (E), the percentage of p24-positive cells (F), the level of hBST-2 on the surface of whole cells (G), and the level of hBST-2 on the surface of p24-positive cells (H) following infection with either wild-type (opened circles with line) or Vpu-deficient (filled circles in gray with broken line) HIV-1 were sequentially measured. The amount of p24 in the culture supernatant was quantified by p24 ELISA, and the other data were obtained by flow cytometry as described in the Materials and Methods. Gray line in panel G indicates MFI of surface hBST-2 on mock-infected cells. All experiments were performed in triplicate. Asterisks indicate statistical significance (Student's t test, P < 0.05) versus the values of Vpu-deficient HIV-1 at the same time point, and double daggers in panel H indicate statistical significance (Student's t test, P < 0.05) versus the values of wild-type HIV-1 at 24 hours post-infection. Error bars indicate standard deviations.