FIG 7.

Tetherin antagonism by Nef is associated with a loss of antitetherin activity by Vpu. (A and B) The Vpu proteins of HIV-1 group M isolates with tetherin-antagonizing Nefs were tested for their ability to rescue virus release for HIV-1 NL4-3 Δvpu Δnef in HEK293T cells expressing human tetherin by measuring p24 levels in culture supernatants by an antigen capture ELISA (A) and by comparing the amount of p24 in supernatants to the amounts of Vpu and p55 Gag in cell lysates by Western blot analysis (B). Deletion mutants of these Vpu alleles (Δ6-11), removing a 6-amino-acid insertion common among subtype C isolates, were also tested. The error bars represent the standard deviations of the means for replicate assays, and differences in mean virus release compared to the empty vector control were significant for NL4-3 Vpu (**, P ≤ 0.01) and AM Vpu (*, P ≤ 0.05) by the Kruskal-Wallis test with adjustment for multiple comparisons. (C) Alignment of the amino acid sequences of the Vpu proteins in panel A to NL4-3 Vpu. Dots indicate amino acid identity, dashes indicate gaps, and residues involved in tetherin antagonism are shaded. Vpu polymorphisms corresponding to positions previously shown to be important for tetherin antagonism are highlighted in blue, and residues corresponding to a 6-amino-acid insertion common among subtype C Vpu alleles are shaded in green.