Figure 2

Anti-tetherin activity in group O proviral clone pCMO2.5. (A) Five μg of group M (pNL4-3) or group O (pCMO2.5) proviral clones were transfected into HeLa cells, and cell lysates and supernatants harvested and analyzed by Western blotting with an anti-p24 antibody. The percent virus release was calculated as the ratio of p24-reacting bands in the supernatants relative to the cell lysates, and normalized to 100% for the virus release from pNL4-3, for n = 2 independent experiments. (B) HeLa cells were transfected with 500 ng of a GFP expression plasmid alone (red), or together with 2 μg of either an expression plasmid for group M Vpu, or 5 μg of the proviral clones pNL4-3 or pCMO2.5 (blue). Cells were stained with an anti-tetherin antibody and analyzed for cell surface tetherin expression by FACS. The histograms show relative cell numbers (% of maximum) vs. tetherin expression (fluorescence intensity of APC) in cells gated for GFP expression; graph shows mean MFI in GFP-positive populations for n = 3 independent experiments, p < 0.01 (**). (C) Human (Hum), chimpanzee (Cpz), macaque (Mac), or a chimeric human tetherin, H(+5), containing an insert from Cpz-tetherin in the cytoplasmic tail, were transiently expressed in 293A cells, together with proviral clones pNL4-3, pNL4-3ΔVpu or pCMO2.5. The percent virus release was calculated as described above and made relative to the no tetherin control for each virus, for n = 4 independent experiments. The Vpu antisera used does not cross-react with the group O protein.