FIG. 4.

CD4 downregulation activity and subcellular localization of Nef-Vpr. (A) Cell surface CD4 expression was analyzed with cells coexpressing CD4 or CD4ΔCT along with Nef variants or Vpr as indicated. Cells were also cotransfected with a plasmid encoding GFP to gate the analysis of cell surface CD4 expression on transfected cells. Dot plots of cells stained with a CD4-specific antibody are shown. (B) Quantification of the CD4 downregulation activity of Nef variants measured as described for panel A. (C) Cellular distribution of Nef, Nef-Vpr, and Vpr. HeLa cells were grown on coverslips and transfected with the indicated HA-tagged constructs. Twenty-four hours posttransfection, cells were permeabilized and stained with an HA-specific antibody and an Alexa488-coupled secondary antibody. Coverslips were transferred to slides in mounting medium supplemented with DAPI and samples were analyzed by immunofluorescence microscopy followed by deconvolution as described in Materials and Methods.