Pronase treatment removes non-internalized virus particles from the cell surface. MT-2 cells were infected with (a, c) HIVGFP-Vpror (b, d) HIVΔenvGFP-Vpr for 2 h at 17°C to allow binding of the virions to the cells. Afterwards the cells were washed to remove unbound virus particles and incubated for 20 min at 37°C to allow virus entry into the cells. The samples were then split and half of the cells were incubated with PBS (a-b) while the other half was treated with pronase (c-d) to remove non-internalized virus particles. Subsequently, all samples were fixed, counterstained, mounted and visualized by widefield microscopy followed by deconvolution. GFP is shown in green and nuclei in blue. The provided images were derived from a volume compression of a z stack of 28 images taken at a 0.3-μm step size. Scale bar, 5 μm. Images are representative of 3 independent experiments.