Vpr transactivation of integration-defective HIV-1 is partially dependent on cis-enhancer sequences. HeLa cells (2 × 10⁴ cells) were coinfected with integrase-defective viral vectors as indicated and HR-eGFP or HR-Vpr (200 ng of p24 antigen). HIV vector indicates infection with HIV-based vector expressing luciferase under the control of an intact HIV-1 LTR (HIV LTR) (MOI of 0.1, using 25 ng of p24 antigen), an HIV LTR with a mutation in the NF-κB (ΔNF-κB) or the Sp1 (ΔSp1) or both the NF-κB and Sp1 (ΔNF-κB/ΔSP1) binding sites, or an HIV LTR containing a 400-bp deletion in U3 (sin18) (MOI of 1.0, using 200 ng of p24 antigen). MLV vector indicates infection with MLV-based vector expressing luciferase under the control of an internal HIV LTR. Luciferase activity was analyzed at day 2 postinfection and normalized by micrograms of total protein. The induction change (n-fold) by Vpr is calculated as the level of luciferase activity in the HR-Vpr-infected cultures relative to that of the HR-eGFP-infected cells, shown as the mean ± standard deviation of three separate wells. This is a representative experiment from five independent experiments.

Tat is dispensable for Vpr-induced expression from integration-defective HIV. HeLa or HeLa-tat cells (2 × 10⁴) were infected at an MOI of 0.1 with vpr-deficient, tat-deficient, luciferase-expressing HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E (25 ng of p24 antigen) and coinfected at an MOI of 1.0 with HR-eGFP or HR-Vpr (200 ng of p24 antigen). Luciferase activity was analyzed at day 2 postinfection. The (n-fold) change in induction in the absence of Vpr and in the presence of Tat was calculated by coinfection with HR-eGFP and HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E in HeLa cells relative to that of HeLa-tat cells. The (n-fold) change in induction in the absence of Tat and in the presence of Vpr was calculated by coinfection of HeLa cells with HR-eGFP and HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E relative to that of the coinfection of HeLa cells with HR-Vpr and HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E. The effect of both Vpr and Tat was calculated by coinfection of HeLa cells with HR-eGFP and HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E relative to that of the coinfection of HeLa-tat cells with HR-Vpr and HIV Vpr⁻/Tat⁻/IN⁺ or HIV Vpr⁻/Tat⁻/D64E.

Vpr-mediated expression of Nef from HIV IN⁻ results in CD4 downregulation. SupT1 cells (1 × 10⁵) were infected at an MOI of 0.2 with 200 ng of p24 antigen of wild-type or integrase-defective HIV expressing Vpr (HIV IN⁺/Vpr⁺ or HIV D64E/Vpr⁺), wild-type or integrase-defective HIV deficient in Vpr (HIV IN⁺/Vpr⁻ or HIV D64E/Vpr⁻) or wild-type or integrase-defective HIV-1 containing Vpr within the virions (HIV IN⁺/Vpr in trans or HIV D64E/Vpr in trans), as indicated. Cells were analyzed for CD4 expression at day 2 postinfection by monoclonal antibody staining and flow cytometry. Percentages of the CD4-positive and CD4-negative cells are shown for each sample. The x axis indicates CD4 staining, and the y axis indicates side scatter (SSC).

Tat is expressed from integrase-defective HIV-1, and the activity is not affected by Vpr. HeLa cells (2 × 10⁵) were transfected with pGL3-LTR luc and plated in 24-well plates. At 24 h posttransfection, cells (4 × 10⁴) were infected at an MOI of 0.5 with 200 ng of p24 antigen of HIV IN⁺/Vpr⁺, HIV IN⁺/Vpr⁻, HIV D64E/Vpr⁺, or HIV D64E/Vpr⁻, as indicated. At day 2 postinfection, cells were harvested and subjected to luciferase analysis. The (n-fold) change in transactivation by Tat is calculated as the level of luciferase activity in the transfected infected cultures relative to that in the transfected uninfected cultures.

Vpr increases expression of the Nef protein from IN⁻ HIV. SupT1 cells (1 × 10⁵) were infected at an MOI of 0.2 with 200 ng of p24 antigen of wild-type or integrase-defective HIV expressing Vpr (HIV IN⁺/Vpr⁺ or HIV D64E/Vpr⁺; lanes 1 and 4), wild-type or integrase-defective HIV deficient in Vpr (HIV IN⁺/Vpr⁻ or HIV D64E/Vpr⁻; lanes 2 and 5), or wild-type or integrase-defective HIV-1 containing Vpr within the virions (HIV IN⁺/Vpr in trans and HIV D64E/Vpr in trans; lanes 3 and 6) and analyzed 2 days postinfection by Western blotting analysis for the presence of Nef. The arrow indicates the protein band corresponding to Nef. Lysates from cells infected with viruses expressing luciferase in place of nef, IN⁺/Luc, and D64E/Luc (lanes 7 to 10) were included to indicate the specificity of the anti-Nef antibody.