FIGURE 5.

TM domain of BST-2 is specifically recognized by Vpu. A, shown is the predicted topology of BST-2 chimeric proteins. The CT, TM, and extracellular (EC) domains of BST-2 are represented in white, and the CT and TM domains of TfR are in black. Plasma membranes are depicted in gray. B, expressions of WT and chimeric BST-2 proteins were confirmed by immunoblotting using the anti-BST-2 polyclonal antibody, which recognizes the extracellular domain. C–E, the TM domain of BST-2 is required for interaction with Vpu and thereby determines the sensitivity to Vpu. C, cell-surface expressions of chimeric BST-2 are shown. 293T cells transiently expressing EGFP, with (lower panels) or without Vpu (upper panels) together with FLAG-tagged BST-2 WT, -TfRct/tm, -TfRct, or TfRtm were stained for cell-surface BST-2 using the anti-FLAG monoclonal antibody and analyzed by two-color flow cytometry. D, shown is the interaction of Vpu with the BST-2 TM domain. Immunoprecipitations (IP) were performed as described in the legend for Fig. 1A, right panels, except that chimeric BST-2 proteins were analyzed. Aliquots of the cell lysates were also analyzed by immunoblotting in parallel for BST-2 (middle panels) and Vpu (lower panels). Arrows indicate the specific bands corresponding to the chimeric BST-2 proteins. Asterisks indicate the positions of immunoglobulin G light chains. E, inhibition of virion release by BST-2 chimeric proteins and their sensitivity to Vpu is shown. The assay was performed as described in Fig. 4D, except that chimeric BST-2 proteins were analyzed. Data are presented as a percentage of the amount of Vpu (−) virion release in the absence of BST-2 and are shown as the mean ± S.D.