Fig. 6

Vpr released from HEK 293T producing cells and Magic5B HIV-1 infected cells can be taken up by Daudi B-cells. a VLPs able to drive the constitutive expression of Vpr by delivery of the pHR-Vpr (Vpr⁺) lentiviral expression cassette or control VLPs containing a pHR-ΔVpr cassette were used to transduce HEK 293T. After 72 h, transduction efficiency was confirmed by the expression of GFP. b Cells were then washed and co-cultured with Daudi B-cells suspended in culture hanging inserts. After an additional 72 h, Daudi cells were collected, immobilized on poly(l-lysine) coated cover slips, formalin-fixed and labeled with DAPI and anti-Vpr antibodies to visualize nuclei and Vpr content, respectively. For each condition, whole cell extracts of producer HEK 293T cells (c) and of target Daudi cells (d) were analyzed for UNG2 content by immunoblot using αtubulin as a loading control. Lanes were cropped from blot membranes serially probed with anti-UNG2 then with anti-αTubulin antibodies (see Supplementary dataset). Uracil DNA glycosylase activity in the lysate was determined in the absence (NT) or presence of the UNG inhibitor UGI (0.2 U). Values are the means of triplicate experiments ± SD. Statistical significance was determined using the ANOVA test. Vpr content was determined by immunoprecipitation using anti-Vpr antibodies. Vpr was visualized by extending exposition time as specified in Additional file 2: Supplementary dataset 2. e Daudi cells maintained in hanging cell inserts were left in contact for the indicated time with MAGIC5B cells previously infected with HIV-1 wt or ΔVpr at MOI of 10. Target Daudi and producer MAGICB cell populations were harvested and whole cell extracts were prepared for immunoblot analysis of their UNG2 levels using αtubulin as a loading control. Lanes were cropped from blot membranes serially probed with anti-UNG2 then with anti-αTubulin antibodies (see Additional file 2: Supplementary dataset 2)