Subcellular localization of mutant forms of Vpr with specific substitutions. (A) Replacement of isoleucine and leucine residues within predicted α-helical domains αH1 and αH2 (represented by shaded boxes) of Vpr. The positions of the substituted amino acids are numbered, and amino acids are shown in the single-letter code. Hatched boxes represent the Flag tag. (B) Transfection of HeLa cells with pME18Neo, which encodes Flag-tagged wild-type or mutant Vpr, or with control vector pME18Neo-Flag. At 24 h after transfection, cells were subjected to immunofluorescence staining as described in the legend to FIG. 1 and analyzed by confocal laser scanning microscopy with the focal plane near the center of each nucleus. Bar, 20 μm.