Figure 4.

HIV-1 AD8 Nef antagonises the long (l-) isoform of tetherin and moderately increases virus release. (A) HEK 293 cells expressing s- or l-tetherin were transfected with AD8 Nef or a control plasmid for two days, and cell surface tetherin levels were quantified by flow cytometry. Mock-transfected cells were immunolabelled with anti-HRP as a staining control. To discriminate intact cells from debris, all cells were also stained and gated for cell surface CD81. The left-hand and central panels show the result from a representative experiment; the right-hand panel shows the average relative tetherin levels from three independent experiments ± SEM with relevant p-values; (B) HEK 293T cells were transfected with 400 ng of HIV-1 AD8 proviral DNA and increasing amounts of l- or s-tetherin-encoding plasmids. Virus and corresponding cells were harvested two days post-transfection, and all lysates were analysed by western blotting. Virus p24 Gag levels were normalised to cellular p55 Gag. The graphs show the average relative virus p24 Gag levels from at least four independent experiments ± SEM with relevant p-values. Tetherin expression was confirmed, as shown in Figure S1A.