FIG 7.

Counteraction of human tetherin by full-length SIVsmm/mac and HIV-2 Env proteins. (A) HEK293T cells were transfected with pCAGGS expression plasmids for the indicated Env proteins. Two days posttransfection, cells were lysed and Env expression was monitored by Western blotting using an anti-HIV-2 Env antiserum. GAPDH served as a loading control. (B) Env functionality was determined by infection of TZM-bl reporter cells with pseudotyped env-deficient mutants of HIV-1 M NL4-3 or HIV-2 AB 7312A. One representative experiment, measured in triplicates ± SEM, is shown. (C) Tetherin surface levels of HeLa cells transiently expressing the indicated Env proteins and an env-deficient mutant of HIV-2 7312A. Flow cytometry was performed 2 days posttransfection, and the mean fluorescence intensity of the tetherin staining in Env-expressing cells was normalized to that of the empty vector control. Mean values from 3 to 6 independent experiments ± SEM are shown. (D and E) Relative p27 release from tetherin knockdown HeLa cells and the respective control cell line expressing scrambled short hairpin RNA (shRNA). HeLa cells were cotransfected with an env-deficient mutant of HIV-2 7312A and expression plasmids for the indicated Env proteins. Two days posttransfection, p27 concentrations were determined by ELISA, and relative p27 release was calculated by normalizing the amount of cell-free p27 to that of cell-associated p27. Mean values from 3 to 8 independent experiments ± SEM are shown. In panel D, values were normalized to control cells expressing tetherin but no antagonist. In panel E, the vector controls of both tetherin-expressing and tetherin knockdown cells was set to 100%. Asterisks in panels C, D, and E indicate statistically significant differences compared to the vector control (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (F) Flow cytometric analysis of HeLa cells stably expressing scrambled (ctrl.) or tetherin-specific (teth.) shRNA. Cells were either stained with a tetherin-specific APC-conjugated antibody (anti-teth.) or the respective isotype control. (G) The top panel illustrates the correlation of tetherin surface levels shown in panel C and relative p27 release shown in panel E. In the lower panel, p27 release from HeLa cells was correlated with p27 release from PBMCs (Fig. 6E). As fusion of SIVtan Env to HIV-2 7312A resulted in a loss of anti-tetherin activity, correlation with (gray) and without (black) SIVtan Env is shown.