Fig. 3

An alanine face on tetherin is required for sensitivity to HIV-2 Env. a HIV-1 VLPs were produced in the presence of wild-type tetherin, or mutant tetherins with single amino acid changes at position 100, together with ROD10 Env or HIV-1 Vpu. VLP release was analyzed by Western blotting and results normalized to the no tetherin control for n = 3 independent experiments, p < 0.05 (*). b The ability of ROD10 Env to bind tetherin mutant A100D was investigated by co-IP in 293T cells, using a GFP-tagged ROD10 Env. c The tetherin sequence from amino acids 95 through 108 is shown above the solved 3D structure of a dimer of the protein’s extracellular domain [17], created using PyMOL software (Schrödinger LLC). Four conserved alanine residues are boxed, and highlighted in black on one monomer in the 3D structure. d Each of the four conserved alanines were individually mutated to aspartic acid, and the ability of the resulting tetherin mutants to restrict VLP release, and be counteracted by Vpu or ROD10 Env, was assessed as previously described. Results were normalized to the no tetherin control, for n = 4 independent experiments, p < 0.05 (*).