Figure 6

Role of Alanine-18 in tetherin antagonism by M-O chimeric Vpu proteins. (A) Schematic of TM domains from M14O and M18O, highlighting location of alanine-18, and configuration of M14O-N18A. (B) Sequence alignment of Vpu TM domains from indicated viruses, with numbering based on group M protein. Alanine residues that are conserved in the functional group M and N Vpu proteins, but are absent in the non-functional group O and P proteins, are labeled in red; non-aromatic hydrophobic residues are labeled in green. Also shown is the 3-D structure of the group M Vpu TM domain (residues 7 to 25 from isolate BH10) [57], created using PyMOL software (Schrödinger LLC), with the conserved alanine residues highlighted in red. (C) Effects of indicated Vpu proteins on HIV-1 VLP release from HeLa cells, measured as previously described, p < 0.01 (**). (D) Effects of indicated Vpu proteins on cell surface tetherin in HeLa cell, measured as previously described, p < 0.05 (*) or p < 0.01 (**). (E) Subcellular localization of M14O and M14O-N18A proteins in HeLa cells, detected by confocal microscopy. Vpu proteins were visualized using anti-FLAG antibody (green), and the TGN (red) was detected with specific antisera. (F) The degree of co-localization of Vpu proteins with the TGN marker was calculated using the Pearson coefficient.