VpxVLP, exogenous deoxynucleosides and SAMHD1 KD increase HIV-1 infection of MDDC. MDDCs were stimulated with 100 ng/ml LPS for 18 hrs or not (ctrl), then treated for 2 hrs with either 2.5 mM deoxynucleosides (dN), SIV Vpx-VLPs, or ΔVpx-VLPs, and finally challenged with a VSV G pesudotyped HIV-1 reporter vector (NL4-3 GFP). 3 days later, > 0.5 x 105 out of 0.5 x 106 transduced cells were assessed for GFP expression by flow cytometry, and the detection limit was set to 0.02% (<10 events). Data is displayed as percent GFP-positive cells (A) or the higest dilution displayed as fold-change compared to the levels obtained with ΔVpx-VLPs (B). MDDCs were treated for 2 hrs with 1 μg/ml MG132 or DMSO prior to addition of Vpx-VLPs or 2.5 mM deoxynucleosides and challenged with HIV-1 reporter vector 2 hrs later (C). 0.5 x 105 MDDCs were transfected twice with 20 nM siRNA targeting SAMHD1. 6 hrs after the second transfection cells were stimulated for 18 hrs with 100 ng/ml LPS. Cells were then treated for 2 hrs with Vpx-VLPs or ΔVpx-VLPs, challenged with NL4-3 GFP, and analyzed by FACS three days later (D). 0.5 x 106 cells were harvested at the time of challenge with GFP-reporter virus to check SAMHD1 protein levels by western blot (E). All experiments here were repeated on at least three separate occasions with similar results using cells from separate, random, healthy blood donors.