Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.