Figure 2
Vpx degrades SAMHD1 in the presence of the antiviral state but does not increase nucleotide levels. MDDCs were stimulated with 100 ng/ml LPS for 18 hrs and treated with SIVMAC VLPs containing Vpx, VpxQ76A or no Vpx (ΔVpx) for 24 hrs. 0.8 x 105 cells were harvested for SAMHD1 western blot (A). SAMHD1 protein levels were measured and normalized to the actin signal. The ΔVpx sample was set to 100% (B). 2 x 106 were harvested for nucleotide extraction and measurement of intracellular deoxyadenosine concentration (C). 0.5 x 106 were challenged with NL4-3 GFP after 2 hrs of VLP treatment in the presence of LPS (D) or absence (E) and 0.5 x 105 were assessed for GFP expression by flow cytometry 3 days later. 1.6 x 106 cells were treated with either 1 μM MLN4924 or DMSO for 2 hrs prior to Vpx-VLPs or ΔVpx-VLPs addition. 24 hrs later cells were harvested for SAMHD1 western blot (F) or deoxyadenosine concentration measurement for one sample per condition (G).