Fig. 1

Characterization of the Vpr/UNG2/RPA32 molecular complex. a, b In vitro binding analyses of Vpr/UNG2/RPA32 interactions. 293T cells were cotransfected with plasmids for expression of HA-tagged forms of Vpr, UNG2 and RPA32. Lysates from transfected cells were then incubated with 5 µg of GST, GST-UNG2 (a) or GST-RPA32 (b) immobilized on GSH-Sepharose beads. Bound proteins were resolved by SDS-PAGE and analyzed by Western blot with anti-HA and anti-β-actin antibodies. Equal amount of cell lysate proteins from transfected cells was run as control on the left panels. c Co-immunoprecipitation of the Vpr/UNG2/RPA32 complex. 293T cells were tranfected with the HA-Vpr expression plasmid or the control plasmid (mock). Cells were lyzed 48 h later and Vpr was precipitated with anti-HA antibody. Immunoprecipitates (right panels) and cell lysates (left panels) were then analyzed by Western blotting with anti-HA, anti-UNG2, anti-RPA32 and anti-β-actin antibodies. d Schematic representation of UNG2 showing the interaction domains with Vpr and the RPA32 (p32) subunit of the RPA complex. The 231–234 WxxF motif of UNG2 (indicated in blue) interacts with Vpr [35] while the N-terminal part of UNG2 encompassing amino-acids 73–84 (in green) contains determinants for RPA32 binding [16, 18, 19]