Figure 6.

HIV-1 AD8 Nef antagonises tetherin by enhanced internalisation and perinuclear accumulation. (A) HEK cells expressing s- or l-tetherin were transfected with AD8 Nef or a control plasmid for two days then fixed and stained for intracellular tetherin. Single confocal sections from a representative experiment are shown. Scale bars = 15 μm. The graph shows accumulation of tetherin in a TGN46-positive perinuclear region in AD8 Nef-expressing cells. All values are normalised to mock-transfected cells, so that a value of 1 indicates no tetherin enrichment. The data show the average of three independent experiments ± SEM with relevant p-values; (B) AD8 Nef- or mock-transfected HeLa cells were immunolabelled for tetherin on ice and shifted to 37 °C for various times. Residual cell surface tetherin antibody was then revealed by immunostaining on ice with a fluorescent secondary antibody. Cells were fixed, permeabilised, immunostained for Nef and analysed by flow cytometry. The three left-hand panels show the result from a representative experiment for mock-transfected cells or AD8 Nef-transfected cells that did (Nef-pos) or did not (Nef-neg) express Nef. The right-hand panel shows the average endocytic rates from four independent experiments ± SEM during the first 30 min at 37 °C with relevant p-values. Endocytic rates were derived from the regression analysis shown in Figure S2B.