Immunofluorescent Localization of HIV-1 Gag Expressed from a Proviral Construct and Effects of Vpu and Endocytosis Inhibitors

<div><p>(A) HeLa cells (left and center panels) or HOS cells (right panel) were transfected with NL4.3 or NL4.3delVpu proviral plasmids, as indicated. Gag localization was determined by immunofluorescence using an α-CA antibody as described in Materials and Methods.</p><p>(B) HeLa cells expressing Gag from the NL4.3delVpu proviral plasmid, and either DN (S34N, upper panels) or constitutively active (Q79L center and lower panels) mutants of CherryFP-Rab5a (red), were fixed and subjected to immunofluorescence to detect HIV-1 Gag (green) at 20 h post-transfection. White bars in the micrographs indicate a distance of 10 μm. The lower three images in (B) show expanded views of a portion of the overlay panels (indicated by the dashed squares) to exemplify how HIV-1 Gag and the CherryRab5a proteins are juxtaposed, Note that Gag sometimes appears to be within the lumen of the CherryFP-Rab5a(Q79L)–marked endosomes.</p><p>(C) The numbers of HIV-1 Gag–expressing cells in ten fields exhibiting PM Gag immunofluorescence only (black bars) or additional intracellular accumulations (white bars) after transfection with NL4.3 or NL4.3delVpu proviral plasmids in the presence of the indicated co-expressed cellular proteins was counted, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020039#ppat-0020039-g002" target="_blank">Figure 2</a>C. Data are plotted as a percentage of the total number of cells with visible Gag accumulations.</p>