FIG 2.

Tetherin's sensitivity to Vpu is dependent on serines, a threonine, and tyrosines. (A) Cytoplasmic tail amino acid sequences of mutant tetherins (THNs) used to determine the residues required for sensitivity to HIV-1 NL4.3 Vpu. (B and C) 293T cells were transfected with increasing amounts of different mutant tetherins and a HIV-1 NL4.3 WT or ΔVpu proviral plasmid. (B) At 48 h posttransfection, infectious virus release was determined with TZM-bl HeLa reporter cells. Error bars represent standard deviations of the means of three independent experiments. Solid lines, NL4.3 WT provirus; dashed lines, NL4.3 ΔVpu provirus. RLU, relative light units. (C) Cell lysates and pelleted viral supernatants from panel B were subjected to SDS-PAGE and analyzed by Western blotting for Hsp90, tetherin, and p24 CA. The percentages of p24 release into the supernatant indicated at the bottom are relative to the release of NL4.3 WT or ΔVpu provirus in the absence of tetherin. (D) 293 cells stably expressing the mutant tetherins were analyzed for surface tetherin expression and are shown in comparison to 293 cells expressing L-tetherin (solid gray line) and empty-vector 293 cells (dashed gray line).