Fig. 4

Substitution of an alanine face in art-tetherin. a Schematic representation of tetherin, artificial tetherin (art-tetherin), and art-tetherin derivatives with insertions of the alanine face (amino acids 95–108), at position 114–117 of art-tetherin. b HIV-1 VLPs were produced in 293A cells as previously described in the presence of ROD10 Env or the control Ebola GP (EboGP) expression plasmids. Additionally, art-tetherin (500 ng), insert 114, or insert 116 (1 μg each) were included and VLP release was analyzed as previously described, with results normalized to the no tetherin control, for n = 3 independent experiments, p < 0.05 (*). c 293T cells were co-transfected with the indicated artificial-tetherin variants and GFP-tagged ROD10 Env. Cells were lysed 24 h later and GFP-tagged proteins immunoprecipitated (IP) with anti-GFP MicroBeads, followed by Western blotting of the input lysates (1%) and IP products using anti-Env, anti-GFP, anti-HA (for art-tetherin) or anti-tetherin antibodies.