E59, L63 and V64 in the second alpha helix of the Vpu cytoplasmic tail are required to efficiently counteract tetherin.

<p>(<b>A</b>) Schematic representation of alanine scan mutagenesis in the second alpha helix of the codon-optimized HIV-1 NL4.3 Vpu protein. (<b>B</b>) 293T cells were transfected with NL4.3 wt or NL4.3 delVpu proviruses in combination with tetherin and the indicated pCR3.1 Vpu-HA expression vectors. 48 h post transfection, cell lysates and pelleted supernatant virions were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu-HA and Hsp90 serving as loading control, and analyzed by LiCor quantitative imager. (<b>C</b>) Viral supernatants from <b>B</b> were assayed for infectivity using HeLa-TZMbl reporter cells. Infectious virus release is plotted on a log scale as β-galactosidase activity in relative light units (RLU). Error bars represent standard deviations of the means of three independent experiments. (<b>D</b>) E59A, L63A and V64A mutations were inserted into the <i>vpu</i> gene of the NL4.3 provirus referred to as NL4.3 Vpu ELV. 293T cells were transfected with NL4.3 wt, NL4.3 delVpu or NL4.3 Vpu ELV proviral plasmids together with increasing doses of tetherin expression vector. The resulting infectivity was determined as in <b>C</b>, error bars represent standard deviations of the means of three independent experiments. (<b>E</b>) Cell lysates and pelleted viral supernatants from 0 ng and 100 ng tetherin input from <b>D</b> were subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA and Hsp90, and analyzed by LiCor quantitative imager.