FIG. 1.

Interaction of tetherin and Vpu via TM domain. (A) Schematic diagram of KGN or KGC fusion protein used in this study. KGN- or KGC-peptide tag was fused in frame to the N terminus of hu-tetherin, mo-tetherin, or mutant proteins. KGN-tag was fused in frame to the C terminus of Vpu or its mutant proteins. The L indicates a linker sequence inserted between KG fragments and proteins of interest. Human, white; mouse, black; the broken line indicates deletion residues. (B) Tetherin-Vpu complex detected by BiFC. HEK 293 cells were transfected with the indicated DNA and examined by confocal microscopy (upper) and immunoblot analysis (lower). (C) BiFC-expressing cells were stained with markers specific for each organelle and imaged by confocal microscopy. Bar, 10 μm. The ratios of the merged area between BiFC (green) and organelles (red) were quantified (right columns) for more than 100 cells. (D) Quantitative BiFC assay. The KGN-Vpu-expressing cells were transfected with pKGC-hu-tetherin (0, 100, 200, or 400 ng) or a control [pKGCstop, 400 ng] and analyzed by flow cytometry (upper) and immunoblot analysis (lower). Relative MFI values are defined as the MFI of tetherin plasmid-transfected cells minus the MFI of untransfected cells, and results represent the means from three independent experiments plus standard deviations.