FIGURE 6.

FLNa depletion inhibits the activity of HIV-1 Vpu to enhance virus release. A and B, A7 cells (A) or M2 cells (B) were cotransfected with HIV-1 Gag-Pol and tetherin expression plasmids at a ratio of 1:1, followed by Western blotting analysis of cell lysates and pelleted VLP lysates using HIV Ig. The right panels represent relative particle release efficiency (**, p < 0.01; ***, p < 0.001). Relative particle release efficiency was determined as p24 levels in the supernatant as a fraction of total p24 levels (supernatant plus cell lysates) and normalized to the particle release efficiency in A7 (A) or M2 (B) cells expressing HIV-1 Gag-Pol alone. The results shown represent the mean ± S.D. (n = 3). C, A7 cells were cotransfected with Vpu-defective NL4–3 proviral plasmids (NL4–3/Udel) and either empty pcDNA3.1 vectors or HIV-1 Vpu expression plasmids, followed by immunoblot analysis of cell lysates (anti-Vpu antibodies, HIV Ig, and anti-actin antibodies) and virion lysates (HIV Ig). The right panel represents relative particle release efficiency (*, p < 0.1). Relative virus release efficiency was determined as p24 levels in the supernatant as a fraction of total p24 levels (supernatant plus cell lysates) and normalized to the virus release efficiency in A7 cells only expressing NL4–3/Udel. The results shown represent the mean ± S.D. (n = 3). D, M2 cells were cotransfected with NL4–3/Udel and HIV-1 Vpu expression plasmids at different ratios (from 1:0 to 1:2), followed by Western blotting analysis of cell lysates (anti-Vpu antibodies, HIV Ig, and anti-actin antibodies) and virion lysates (HIV Ig). The right panel represents relative particle release efficiency (ns, no significance). Relative virus release efficiency was determined as p24 levels in the supernatant as a fraction of total p24 levels (supernatant plus cell lysates) and normalized to virus release efficiency in M2 cells only expressing NL4–3/Udel. The results shown represent the mean ± S.D. (n = 3).