Vpx and Vpr have toggled throughout evolution in their requirement for the N- or the C-terminus of SAMHD1.

<p>(A) 293T cells were transfected with HA-SAMHD1, either WT or ΔC (left panel), or WT or chimeric Human-Rhesus SAMHD1 (consisting of residues 1–114 of human SAMHD1 and 115–626 of rhesus SAMHD1, right panels), plus or minus FLAG-Vpr, and degradation was measured by western blotting as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1</a>. Rhesus SAMHD1 was used as all Vpr tested herein can degrade this SAMHD1. Human SAMHD1 was used in N-terminal chimeras as it contains multiple non-synonymous changes compared to other primate SAMHD1. (B) <i>In vitro</i> ubiquitylation of WT, ΔC, ΔN, and ΔN/ΔC DeBrazza's SAMHD1 (consisting of amino acids 115–595, schematic representation in top panels), in the presence or absence of SIVdeb Vpr, SIVmac Vpx, or SIVrcm Vpx. CRL4 and CRL4-DCAF1 alone were used as controls. Experiment preformed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g004" target="_blank">Figure 4B</a> and ubiquitylation of each SAMHD1 construct was analyzed by western blotting. Timepoints shown are 0, 15, 30, and 60 minute incubations. (C) Schematic Vpx/Vpr phylogenetic tree as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1A</a> and SAMHD1 diagrams depicting dependence of Vpx/Vpr on the N- or C-terminus of its autologous SAMHD1. Red indicates strong dependence based on all assays. Light pink indicates slight dependence based on co-IP data. Magenta indicates intermediate dependence.