Figure 6. AP180c inhibits Vpu-mediated tetherin antagonism but AP-1, AP-2, AP-3 and retromer are dispensable.

(A) 293T/tetherin were transfected with NL4.3 wt or NL4.3 delVpu proviral plasmids in combination with either YFP or increasing doses of an AP180c expression vector. 48 h post transfection, cell lysates and pelleted supernatant virions were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu and Hsp90 serving as loading control, and analyzed by LiCor quantitative imager. (B) 293T or 293T/tetherin transfected with pCR3.1 Vpu-YFP with or without AP180c co-expression were fixed and imaged after 48 h (C) 293T cells stably expressing tetherin were transfected twice with pooled control or AP-2μ1 siRNAs and co-transfected with NL4.3 wt, NL4.3 delVpu or GFP expression vectors. Cell lysates and supernatants were analyzed by Western blotting 48 h later as described in Figure 1B. (D) 293T cells stably expressing tetherin were transfected twice with pooled control or siRNA pools against AP-3δ1 and AP-3μ1. 4 h post the second transfection the cells were infected with VSV-G-pseudotyped HIV-1 wt or HIV-1 delVpu virus stock at an MOI of 1. Cell lysates and supernatants were analyzed by Western blotting 48 h later as described in Figure 1B. (E) HeLa cells expressing a doxycycline-inducible shRNA hairpin against AP-1γ1were transfected twice with pooled control or siRNA pools against AP-1γ1. The cells were infected and analyzed as in C. (F) 293T cells stably expressing tetherin were transfected twice with pooled control or siRNA pools against the retromer subunit Vps26. Cells were infected and analyzed as in C. In all siRNA knockdown experiments, the % knockdown of the indicated protein as determined by the relative band intensity in the western is indicated below the blot panel.