Vpr increases viral expression in MDMs through a VprBP-dependent mechanism. A, MDMs were transfected with VprBP or non-targeting siRNAs. After 48 h, MDMs were infected with the CCR5-tropic WT or Vpr-defective (ΔVpr) viruses. Supernatants of the infected cells were analyzed by p24 ELISA. Right: Western blot analysis of samples examined for depletion of VprBP. B, MDMs were infected with WT, ΔVpr, and the mutant (Q65R and R80A) CCR5-tropic viruses and their supernatants were analyzed by p24 ELISA. C, MDMs were infected with the single cycle VSV-G-pseudotyped WT- or ΔVpr-luciferase reporter viruses. After 7 days, luciferase activity was measured. D, MDMs from 8 donors were infected with the single cycle VSV-G pseudotyped WT, ΔVpr, Q65R, and R80A GFP marked viruses. After 4 days, cells were analyzed for expression of GFP. E, one representative experiment as described in 5D. F, primary MDMs were infected with the GFP-marked WT or ΔVpr viruses at an MOI of 1.0. After 7 days, infected GFP-positive cells were sorted and equal numbers of cells were analyzed using Western blot. G and H, MDMs were infected with the single cycle VSV-G pseudotyped WT and ΔVpr GFP-marked viruses. After 4 days, GFP-positive and negative cells were sorted. To determine levels of HIV-1 transcripts, quantitative PCR was performed on the total population (unsorted cells) or the sorted GFP-positive cells.