Vpx-dependent infection of human monocytes correlates with reduced A3A expression levels. A, human monocytes of two donors were infected with 0.5 m.o.i. of PBj1.9 wt or PBj1.9 X2. At days 8 and 15 after infection, reverse transcriptase (RT) concentration in the supernatant was determined. Subsequently, the monocytes were lysed and immunoblotted (IB) with anti-A3A and anti-tubulin antibodies. A3A protein band density was normalized to tubulin protein band density. A3A relative density units (rdu) of wt infected cells were set to 100, showing the relative increase in A3A protein band density in X2-infected cells. B: left, domain structure of Vpx and location of the H82A mutation. H indicates an α-helix, and PR indicates a proline-rich region. Right, 293T cells were transfected with the packaging construct PBj-psi10 and plasmids encoding for vesicular stomatitis virus G and Vpx or H82A-Vpx. Three days after transfection, VLPs were analyzed via immunoblotting with anti-Vpx and anti-p27 antibody. C, 1 day after isolation, primary human monocytes were incubated with different m.o.i. equivalents (MOIeq) of VLPs containing wt Vpx, H82A-Vpx, or empty VLPs. Monocytes were transduced with a m.o.i. = 8 HIV-1 vector particles harboring EGFP as reporter transgene. Monocytes were analyzed via fluorescence-activated cell sorting 5 days later.

Vpx, but not H82A-Vpx, supports degradation of APOBEC3A. A: left, 293T cells were transfected with constant amounts of HA-tagged A3A and increasing amounts of HA-Vpx-encoding plasmid. Proteins were detected by immunoblot (IB) analysis with antibodies against HA tag and β-actin as loading control. Protein band intensity was quantified with densitometry, defining A3A protein levels in the absence of Vpx as 100 relative density units (rdu). Right, 293T cells were transfected and analyzed as the left panel using the H82A-Vpx mutant. B, 293T cells were transfected with HA-A3A and Vpx or H82A-Vpx in a A3A:Vpx DNA ratio of 1:6. Cell lysates were analyzed by immunoblotting using anti-HA and anti-Vpx antibodies. C, U937 monocytic cells were transfected with HA-A3A and HA-Vpx in a DNA ratio of 1:1. Cell lysates were immunoblotted and analyzed with an anti-HA antibody. D, 293T cells were transfected with plasmids encoding HA-A3A and Vpx or empty vector in a DNA ratio of 1:5. 42 h after transfection and 6 h before harvest, cells were treated with 100 μg/ml cycloheximide (CHX) for the indicated period. Western blot analysis was performed as described in B, and protein bands were quantified defining A3A protein levels of cycloheximide-untreated cells as 100 relative density units.

Vpx interacts with A3A, and both proteins co-localize to the same cellular compartment. A, 10 μg of GST or GST-Vpx was incubated with glutathione-coupled Sepharose beads, and after GST pulldown recombinant proteins were separated with SDS-PAGE and stained with Coomassie Blue. B, 10 μg of GST or GST-Vpx was incubated with lysates of 293T cells transiently expressing HA-A3A and glutathione-coupled Sepharose beads. After pulldown, associated proteins were analyzed by immunoblotting (IB) using an anti-HA antibody. C, 293T cells were transiently transfected with HA-A3A and Vpx or H82A-Vpx. After HA-directed immunoprecipitation, proteins were detected with anti-Vpx and anti-HA antibodies in immunoblot analysis. IgG LC indicates the light chain of the IP antibody. D, HeLa cells were transiently transfected with HA-A3A and Vpx or H82A-Vpx. Cell lysates were separated in nuclear (Nuc) and cytoplasmic (Cyt) fractions, and equal protein amounts of the respective fractions were subjected to immunoblot analysis using anti-HA and anti-Vpx antibodies. Anti-laminin A was used as control for a nucleus-specific protein. E, HeLa cells were transfected with HA-A3A and FLAG-Vpx or FLAG-H82A-Vpx. Cellular localization was determined via confocal microscopy using anti-HA and anti-FLAG primary and fluorescent secondary antibodies. Nuclei were stained with DAPI. White bar, 10 μm. F, 293T cells were transfected with HA- and FLAG-tagged Vpx or H82A-Vpx, and FLAG-directed immunoprecipitation was performed. Cell lysates and precipitated proteins were identified with anti-HA and anti-FLAG antibodies in immunoblot analysis.