FIG 4.

Expression of Nef and Nef-mediated counteraction of human tetherin in primary cells. (A) HEK293T cells were transfected with expression plasmids for the indicated C-terminally AU1-tagged Vpu and Nef proteins. Two days posttransfection, cells were lysed and viral protein expression was monitored by Western blotting using an anti-AU1 antibody. GAPDH served as a loading control. (B) Human PBMCs were infected with HIV-1 NL4-3-derived viruses expressing the indicated Nef proteins and analyzed by flow cytometry as described for Fig. 3. The top panel shows mean values from 3 to 12 independent experiments ± SEM. Examples of primary data are shown in the bottom panel. (C) In parallel, p24 contents in the cells and cell culture supernatants were determined by ELISA 3 days postinfection. Relative p24 release from PBMCs was calculated by normalizing the amount of cell-free p24 to that of cell-associated p24. The values of the negative control (vpu- env- nef-) were set to 100%. Mean values from 4 to 12 independent experiments ± SEM are shown on the left. On the right, samples were grouped into SIVsmm/mac and HIV-2 Nefs. n.s., not significant. Asterisks in panels B and C indicate statistically significant differences compared to the negative control (vpu- env- nef-) (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).