Figure 5.

Vpx-mediated SAMHD1 degradation is inhibited by IFNs in THP-1 cells. (A) PMA-differentiated THP-1 cells were either mock treated, or treated with IFN-α or IFN-γ (1000 U/ml) during 24 h, then were incubated with empty or Vpx-containing VLP (X- and X+) for 2 h. Subsequently, cells were transduced with a VSV-G-pseudotyped GFP-encoding HIV-1 virus at MOI 1. The transduction rate was measured 3 days post-transduction (upper panel) and protein expression analyzed (lower panel). The results shown here are representative of three independent experiments. (B) SAMHD1-depleted THP-1 cells (shRNA SAMHD1) or control cells (shRNA scrambled scr) were treated with PMA, then primed with IFN-α (1000 U/ml) for 24 h, before transduction with a HIV-1-Luc reporter virus. The luciferase activity was measured 2 days post-transduction (upper panel) and SAMHD1 expression analyzed (lower panel). ND: not detected. (C) A monoclonal THP-1 cell line stably expressing HA-tagged SAMHD1 as well as wt THP1 cells were differentiated by PMA. Cells were primed with IFN-α (1000 U/ml) for 24 h, before incubation for 2 h with empty or Vpx containing VLP (X- or X+, respectively) and transduced with a HIV-1-Luc reporter virus. The luciferase activity was measured 2 days post-transduction (upper panel). Levels of endogenous and HA-tagged SAMHD1 were analyzed by western blot (lower panel). ND: not detected. (D) PMA-differentiated THP-1 cells were primed or not with IFN-α (10000 U/ml, 24 h). The BlaM-Vpr fusion entry test was then carried out as described in the methods. (E) IFN-α does not modify SAMHD1 nuclear localization. PMA-differentiated THP-1 cells expressing or not HA-SAMHD1 were primed with IFN-α (1000 U/ml) for 24 h. Cells were stained with the indicated antibodies for immunofluorescence (green). Nuclei were stained with DAPI (blue).