Figure 4
From: Lack of adaptation to human tetherin in HIV-1 Group O and P
Characterization of chimeric M-O Vpu proteins. (A) Schematic (not to scale) of major domains in FLAG-tagged chimeric Vpu proteins formed between the functional group M (NL4-3, grey) and non-functional group O (ANT70, black) proteins. Numbers in name indicate junction site and refer to the group M residues. (B) Activity of M-O chimeric Vpu-FLAG proteins against human tetherin in HeLa cells. Relative VLP release was calculated as described previously and is shown for n = 3 independent experiments, p < 0.01 (**). Expression of Vpu-FLAG proteins was confirmed using an anti-FLAG antibody. (C) Vpu-FLAG proteins O and O26M are expressed at lower levels than other Vpu constructs, so increasing amounts of the plasmids were transfected into HeLa cells (range 2 to 6 μg), to confirm that their lack of anti-tetherin activity was not simply due to lower levels of expression. As a control, 2 μg of group M Vpu-FLAG was transfected. (D) Ability of chimeric M-O Vpu-FLAG proteins to remove tetherin from the surface of HeLa cells. Cells were co-transfected with 2 μg of indicated Vpu plasmid and 500 ng of GFP expression plasmid and MFI calculated in the GFP-positive population. Graph shows mean MFI for n = 3 independent experiments, p < 0.01 (**). (E) 293T cells were transfected with HA-tagged tetherin alone (500 ng) or together with the indicated Vpu-FLAG expression plasmids (1 μg), except O and O26M (2 μg). Immunoprecipitation (IP) was performed using anti-HA MicroBeads, followed by Western blot analysis of both input lysates (1%) and immunoprecipitates, using anti-FLAG and anti-tetherin antibodies.