Fig. 4: Integrity of SAMHD1 SIM2 is required for both HIV-1 restriction and K595 SUMOylation.

a Schematic representation of human SAMHD1 (NP_056289.2), showing the position and sequence of the putative SIMs and alignments with corresponding sequences of isoforms from Rhesus macaque (NP_001258571.1), mouse (NP_061339.3, isoform 1), and zebrafish (NP_001153405.1). b Position of SIM2 and SIM3 (green), K595 (red), and E597 (blue) within one protomer of human SAMHD1 tetramer (PDB: 4BZC). c The lysate of differentiated THP1 cells was split in equal aliquots that were incubated with agarose beads coated with either human recombinant SUMO1 (S1) or SUMO2 (S2), or uncoated beads (UC) as control. Proteins from the input and the eluates were separated by migration on a 4–15% SDS-PAGE gel and, next, visualized by immunoblotting using antibodies against SAMHD1. Results of one representative experiment are shown (n = 3). d The lysate of differentiated U937 cells expressing WT or SIM2m SAMHD1 variants was incubated with SUMO2-coated agarose beads and treated as in (c). The band intensities were quantified with ImageJ software. Results of one representative experiment are shown (n = 2). e U937 cells stably expressing the indicated HA-SAMHD1 variants (three independently generated cell lines) were infected with the VSVg/HIV-1∆EnvEGFP virus and analyzed as in Fig. 3b. The infection rate of parental U937 cells was set to 100. Data (mean ± SD) from one representative experiment performed in three technical replicates are shown (n = 5). f HEK 293T cells overexpressing HA-SAMHD1 RKR mutant, Ubc9, and His-SUMO2 were lyzed in denaturing conditions and split in two equal aliquots that were subject to Ni-NTA pull down or affinity purification on HA-matrix beads. Eluted proteins were analyzed as described in Fig. 1a. WCL whole-cell lysate. Results of one representative experiment are shown (n = 2). The red arrowhead highlights the SUMO-conjugated K595 SAMHD1 species. *, nonspecific binding of unmodified SAMHD1 on Ni-NTA beads. Lanes 1–3 are derived from the same blot but were not adjacently loaded.