Fig. 4

Vpx Ser13 phosphorylation strengthens the Vpx–SAMHD1 interaction. a Primary structure of Vpx, showing the SAMHD1-binding (red) and DCAF1-binding (green) motifs¹⁹. Arrow indicates the residue of phospho-Ser13. b Molecular dynamics simulations of Vpx (blue) coupled with peptides derived from SAMHD1 (red) and DCAF1 (gray) in the absence (left) or presence (right) of Vpx Ser13 phosphorylation. c Expanded view of a predicted hydrogen bond between phospho-Vpx and SAMHD1 (shown in an orange line). d, e NanoBRET assays of HEK293 cells expressing NanoLuc-conjugated Vpx and either SAMHD1 (d) or DCAF1 (e) fused with HaloTag. Forty-eight hours after transfection, intracellular NanoBRET signals were calculated. All graphs are presented as a mean ± s.d. (n = 3). ***P < 0.001; ns, not significant, two-tailed unpaired t-test. f–h Vpx-S13A binds SAMHD1 to a lesser extent than wild-type Vpx. HEK293 cells expressing HA-SAMHD1 (f) or HA-DCAF1 (g) and either FLAG-Vpx WT or S13A were lysed; and the resultant lysates were immunoprecipitated with anti-FLAG (f, g) or anti-HA antibodies (h); the precipitants were analyzed by immunoblotting. The numerical values below the blots show the signal intensities of the indicated precipitants, as determined by densitometry. i Vpx-S13A is less capable of poly-ubiquitinating SAMHD1 than wild-type Vpx. HEK293 cells were cotransfected with plasmids encoding HA-SAMHD1, FLAG-Vpx (WT or S13A), and Myc-ubiquitin. The cells were then treated with MG132 for 18 h and harvested. Cell lysates were immunoprecipitated with anti-HA antibody, and the Vpx-induced ubiquitination of SAMHD1 was detected by immunoblotting with anti-Myc antibody. Source data are provided as a Source Data file