HIV-1 Vpr colocalizes with nuclear pores in human cells. (A–C) Optical sections through the nuclei of HeLa cells transfected to express HA–Vpr, stained using indirect immunofluorescence for the HA epitope tag on Vpr (A, red) and antibodies to nuclear lamins (B, green). In the superimposition of staining patterns (C), overlap between the two antigens is visible in yellow. (D–G) Optical section of the nucleus of a HeLa cell expressing Vpr. (E) Staining with mAb 414, which recognizes nuclear pores, outlining the nuclear envelope. (D) Staining for HA epitope tagged Vpr using an antibody to HA, which also outlines the nuclear envelope. (F) The superimposition of nuclear pore staining (green) and Vpr staining (red), visible in yellow, demonstrates the colocalization of the nuclear pore antigens and Vpr. D is a portion of C enlarged; arrows indicate Vpr staining (red) is mostly on the interior, whereas nuclear pore staining (green) is mostly on the exterior faces of the nuclear envelope. Scale bar, 2.5 μm in A–C; 1 μm in D–G.

 Vpr is expressed in yeast cells and colocalizes with the nuclear pore protein Nsp1p in yeast. Wild-type and mutant Vpr were expressed in wild-type yeast cells using a galactose-inducible promoter. Wild-type Vpr (top) colocalizes with the Nsp1p staining, whereas the F34I Vpr mutant (bottom) does not. Vpr was visualized using anti-Vpr polyclonal antibodies; Nsp1p was observed using an affinity-purified polyclonal antisera; DNA was visualized by DAPI staining and cells were viewed with Nomarski optics.

 Vpr interacts with transport factors. Wild-type Vpr and the F34I Vpr mutant were purified from wild-type yeast cells as GST fusions. Samples were resolved by SDS–PAGE and either stained with Coomassie blue dye to demonstrate protein expression (A), and then transferred to nitrocellulose and immunoblotted with anti-importin-α (B) or anti-Nsp1p (C). Importin-α and Nsp1p copurify only with the wild-type Vpr GST fusion and not with the F34I mutant or GST alone. The identical experiment using a GST–importin-β fusion is also shown.

 HIV-1 Vpr localizes to the nuclear envelope but point mutants are defective in localization. Indirect immunofluorescence on HeLa cells transfected with HA–Vpr expression constructs using an antibody to the HA–epitope tag on each Vpr expression construct. Staining for wild-type Vpr (A) is brightest in a ring outlining the nucleus; however, the point mutants, F34I (B) and H71R (C), are found in the nucleoplasm but not on the nuclear envelope. Optical sections are near the middle of each nucleus. Scale bar, 4 μm.

 Wild-type Vpr but not F34I Vpr targets a fusion protein to the nuclear envelope. HeLa cells transfected to express Vpr–β-galactosidase fusion proteins stained with an antibody to β-galactosidase in green, superimposed on fluorescence from DAPI-stained DNA in blue. Arrow in A shows the overlap between β-galactosidase staining in green and DNA staining in blue as aqua, indicating the fusion protein with wild-type Vpr localizes to the nuclear envelope. The lack of aqua overlap in B as indicated by the arrow demonstrates that the F34I mutant fusion protein remains cytoplasmic. Optical sections are near the middle of each nucleus. Scale bar, 2 μm.

 Expression of Vpr causes an mRNA export defect identical to the defect caused by overexpression of importin-β. (A) Importin-α and importin-β were expressed in wild-type yeast cells using a galactose-inducible promoter. Cells were then used for the in situ mRNA export assay. Cells expressing importin-β (top row) show an accumulation of mRNA in the nucleus, whereas cells expressing importin-α (second row) do not and are indistinguishable from wild-type (third row). rat7-1 mutant cells at the restrictive temperature (36°) (bottom row) show the strongest mRNA export defect. (B) Wild-type and mutant Vpr were expressed in wild-type yeast cells and mRNA was visualized using a fluorescence in situ hybridization assay (FISH). Cells expressing wild-type Vpr show a significant nuclear signal, indicating that the mRNA is not being exported properly (top row). Cells expressing the Vpr F34I mutant (bottom row) exhibit staining all over the cell, consistent with the localization of mRNA in wild-type cells. The FITC column shows the mRNA signal; DNA is visualized with DAPI and cells viewed with Nomarski optics. In the FITC column, arrows point to the mRNA signal in the nucleus of cells expressing importin-β or wild-type Vpr from the galactose-inducible promoter. In the DAPI column, arrows point to stained nuclei.

 Effects of wild-type and mutant Vpr on cell cycle progression and macrophage infection. (A) Cell cycle analysis of HeLa cells cotransfected with HA-tagged Vpr constructs and a GFP plasmid. Cells not expressing Vpr G₁ = 51%; G₂ = 5%. Cells expressing Vpr, G₁ = 5%; G₂ = 69%. Cells expressing Vpr F34I, G₁ = 29%; G₂ = 46%. Cells expressing Vpr H71R, G₁ = 42%; G₂ = 8%. Analyses are of GFP positive cells. Cell number is on the y-axis and DNA content as measured by fluorescence intensity of propidium iodide staining is on the x-axis. (B) Vpr mutants defective for nuclear envelope localization cannot sustain HIV-1 macrophage infection. (Right) Primary human macrophages were infected with equivalent infectious units of MA–NLS minus HIV-1 containing either wild-type or mutant vpr. (Left) Transformed human T cells were infected with the same viruses. Media from each sample were collected every 3 days and replaced completely for macrophage infections, whereas T-cell infections were split 1:2. All samples were assayed for p24gag concentration. (x-axis) Days postinfection; (y-axis) p24 pg/ml. (○) HIV-1 MA–NLS⁻, Vpr⁺; (▴) HIV-1 MA–NLS⁻, ΔVpr; (□) HIV-1 MA–NLS⁻, Vpr F341; (♦) HIV-1 MA–NLS⁻, Vpr H71R.