Figure 6

Transduction of MDDCs with lentivectors expressing SIV Vpx or Vpr. Freshly isolated CD14⁺ monocytes from human peripheral blood were treated with either Vpx-VLPs or 2.5 mM deoxynucleosides and transduced with a lentiviral vector expressing GFP. Cells were analyzed over the course of differentiation (day 3 to day 6) for GFP expression by flow cytometry shown as percent GFP-postitve cells (A) or mean fluoresence intensity (B). Monocytes were treated with 2.5 mM deoxynucleosides for 2 hrs and transduced with a lentiviral vector expressing a puromycin-resistance cassette alone (−), or with the indicated SIV_MAC Vpx WT or Vpx Q76A mutant (C), Vpr from SIV_DEB (DEB), Vpr from SIV_MUS (MUS), Vpx from HIV-2 (ROD), Vpx from SIV_RCM (RCM) or the empty vector (Vector) (D). Cells were selected with 1 μg/ml puromycin for 24 hrs at day 3 after transduction. After selection cells were harvested, washed, re-plated and stimulated with 100 ng/ml LPS for 18 hrs. Cells were treated or not with 2.5 mM deoxynucleosides for 2 hrs and challenged with HIV-1-GFP reporter vector. GFP positve cells were assessed for GFP expression by flow cytometry 72 hrs later. Low molecular weight DNA was collected at 24 hrs after challenge and LRT (E) and 2 LTR circles (F) were measured by qPCR.