Figure 2.

HIV-1 AD8 Nef reduces cell surface tetherin levels and tethered virions. (A) AD8 Vpu, Env, or Nef were transfected into HeLa cells for two days. Cell surface tetherin levels were quantified by immunolabelling the restriction factor on ice prior to formaldehyde fixation, cell permeabilisation, immunolabelling for Vpu, Env or Nef, labelling with fluorescent secondary antibodies and flow cytometry. Mock-transfected cells were immunolabelled with anti-VSVG as a control. The left-hand panel shows the result from a representative experiment; the right-hand panel shows the average relative tetherin levels from three independent experiments ± standard error of the mean (SEM); (B) cell surface tetherin levels of VSVG-pseudotyped AD8-infected or uninfected HeLa cells were quantified by flow cytometry. Uninfected cells were immunolabelled with anti-HRP as a staining control. The left-hand panel shows the result from a representative experiment; the right-hand panel shows the average relative tetherin levels from four independent experiments ± SEM with relevant p-values; (C) cell surface Env levels of VSVG-pseudotyped AD8-infected or uninfected HeLa cells were quantified by flow cytometry. The upper panels and lower left-hand panel show the results from a representative experiment; the lower right-hand panel shows the average relative Env levels from four independent experiments ± SEM with relevant p-values.