Functional rescue of Vpu phospho- and trafficking mutants by direct interaction with clathrin.

<p>(A) Schematic representation of Vpu CB chimera constructs. (B) 293T tetherin cells were transfected with NL4.3 ΔVpu proviral plasmid in combination with YFP expression vector and pCR3.1 Vpu, pCR3.1 Vpu CB or Vpu CB mut or Vpu mutants thereof. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. Error bars represent standard deviation of three independent experiments. (C) Cell lysates and pelleted supernatant virions from (B) were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu and HSP90, and analyzed by LiCor quantitative imager. (D) HeLa-TZMbl cells were co-transfected with pCR3.1 Vpu or indicated mutant and a GFP expression vector. Cell-surface tetherin levels were analyzed 48 hours post transfection by flow cytometry in the GFP positive cells. The percentages of tetherin surface expression levels are calculated from median fluorescence intensities.