Figure 7

Alanine-18 is important for group M Vpu anti-tetherin activity. (A) Effect of group M Vpu and the A18H mutant on VLP release from HeLa cells, measured as previously described, p < 0.01 (**). (B) Effect of Vpu on tetherin expression on cell surface of HeLa cells, in the absence (red) or presence (blue) of Vpu, examined as previously described. p < 0.01 (**) (C) Subcellular localization of indicated Vpu constructs in HeLa cells and their effects on tetherin distribution. TGN (top) and ER (bottom) markers are included. Arrows indicate cells expressing Vpu that redistributed tetherin to the TGN. (D) 293T cells were transfected with tetherin alone (500 ng) or together with the indicated Vpu-EGFP expression plasmids (5 μg), where M is the Vpu protein from group M strain HXB2. SIVcpz Vpu-EGFP, which is not active against human tetherin, and a Vpu mutant, A14L, were included as negative controls. Immunoprecipitation (IP) was performed using anti-GFP MicroBeads, followed by Western blot analysis of both input lysates (1%) and immunoprecipitates, using either anti-GFP or anti-tetherin antibodies. Mature glycosylated forms of tetherin run between 25 and 37 kDa (bracketed) and interact specifically with wild-type group M Vpu, while an immature faster running species that is also present in the transfected cells is non-specifically pulled down by all Vpu proteins.