Fig. 6

SUMOylation-deficient SAMHD1 impairs its anti-viral activity. A SUMOylation-deficient SAMHD1 impairs its anti-EBV activity. HEK293 (EBV +) cells were co-transfected with plasmid DNA encoding ZTA (lytic trigger), WT PIAS1, and WT or mutant SAMHD1 as indicated. The relative EBV copy numbers were measured using the qPCR as described in the method. The expression levels of ZTA, SAMHD1 and PIAS1 were monitored by WB. β-actin blot was included as loading controls. Results from three biological replicates are presented. Error bars indicate the standard deviation. *p < 0.05, **p < 0.01. B Akata (EBV +)-SAMHD1-sg1 cells (endogenous SAMHD1 is depleted by CRISPR/Cas9) were used to create cell lines using vector control (pLX304), WT SAMHD1 (pLX-304-V5-SAMHD1), and SUMOylation-deficient SAMHD1 [pLX-304-V5-SAMHD1-(RRR)]. EBV lytic cycle was induced by anti-IgG-mediated B cell receptor cross-linking. The relative EBV copy numbers were measured using the qPCR as described in the method. The expression levels of SAMHD1 were monitored by WB using anti-V5 antibody. β-actin blot was included as loading controls. Results from three biological replicates are presented. Error bars indicate the standard deviation. *p < 0.05, ** p < 0.01. C The relative positions of EBV ZTA, RTA and OriLyt are labeled as indicated. D PIAS1 regulates SAMHD1 association with EBV genome. ChIP-PCR analysis performed on Akata (EBV +) cells showing SAMHD1 binding to ZTA (ZTAp) and RTA (RTAp) promoters and OriLyt region in WT control cells but not PIAS1-knockout (PIAS1-KO) cells. ChIP by a nonspecific IgG was included as a negative control. The expression levels of PIAS1, SAMHD1 and β -actin were monitored by WB. Results from three biological replicates are presented. Error bars indicate the standard deviation. ***p < 0.001. E Hypothesized model. PIAS1 regulates the SUMOylation and viral genome recruitment of SAMHD1 to restrict EBV lytic replication