Figure 3

Anti-tetherin activities in group O and P Nef proteins. (A) Anti-tetherin activity of group O Nef proteins against the indicated tetherins was examined in 293A cells. Graph shows VLP release in the presence of indicated tetherins and Vpu or Nef proteins relative to the baseline levels of release from the tetherin alone controls (-), for n = 3 independent experiments. Group M Vpu, SIVcpz Nef-EGFP and SIVmac239 Nef-EGFP proteins were included as positive controls. Nef proteins were detected using antiserum raised against group M Nef protein that cross-reacts with group O proteins but not SIVmac Nef. Statistical significance is indicated as p < 0.05 (*) or p < 0.01 (**). (B) Human CD4 expression plasmid (1 μg) was transfected into 293A cells, together with 1 μg of the indicated Vpu or Nef plasmids. Group M Vpu and the defective Vpu-S52/56N mutant were included as positive and negative controls for CD4 degradation, respectively. Untagged Nef proteins were probed using anti-group M Nef antiserum and GFP-tagged Nef proteins were detected using anti-GFP antibody. (C) Activity of CMO2.5 Nef and a myristoylation site mutant (CMO2.5 Nef-G2A) against human and H(+5) tetherin, in 293A cells. Group M Vpu and SIVmac Nef were included as positive controls and group M Nef was included as a negative control. (D) Effect of group P Vpu or Nef proteins on HIV-1 VLP release in the presence of different tetherins, measured in 293A cells, as previously described, for n = 2 independent experiments. Vpu and Nef expression was detected using anti-GFP antibody.