Vpu depletes human tetherin but not murine tetherin, which parallels its ability to rescue virion release.

<p>(A) Vpu counteracts human but not murine tetherin antiviral activity. 293T cells were transfected with an HIV-1 provirus either proficient or deficient for the Vpu gene, together with the indicated tetherin constructs. Viral output was then scored by titration of the resulting supernatant on HeLa indicator cells. (B) Vpu depletes human, but not murine, tetherin. Duplicate cell extracts from the above experiment were monitored for tetherin level (as detected with an anti-HA antibody). The viral p55 Gag protein was monitored to exclude variations of transfection efficiency. The depicted tetherin bands correspond to the heterogeneously glycosylated monomer of tetherin of around 30 kDa, but equivalent depletion could be observed for the 60 kDa dimeric form (data not shown). PCNA was used as a loading control. Note that all parts come from the same blot, but the detection of murine tetherin required longer exposure due to its lower expression. (C) Vpu depletes human tetherin in a dose dependent manner, in the absence of other viral components. Increasing doses of a Vpu-expressing plasmid was co-transfected with a Flag-tagged human tetherin in 293T cells (molar ratios of 1∶1 and 2∶1, indicated by + and ++ respectively). Steady state levels of tetherin were monitored by western blot analysis of duplicate cell extracts using an anti-Flag antibody. Transfection levels were assessed with a GFP plasmid, and actin was used as a loading control. All three sections of this figure are representative of five independent experiments performed in duplicate. Sizes of molecular weight markers are shown in kilodaltons.