Fig. 5: Dephosphorylated T592 is not sufficient to render SAMHD1 antivirally active, concomitant SUMOylation of K595 is required.

a Proteins (10 µg total proteins/line) contained in the crude extract of 293 T cells overexpressing HA-SAMHD1 variants were loaded on a 4–15% pre-casted SDS-PAGE gel. Immunoblotting was performed sequentially with an anti-pT592 or anti-HA antibody to detect phosphorylated or total SAMHD1 species, respectively. The band intensities were quantified by densitometry with ImageJ software and the pT592/total SAMHD1 ratio for WT SAMHD1-expressing cells was set to 1. One representative immunoblot is shown, while the quantification data represent the mean ± SD of all the experiments (n ≥ 2). The ability of SAMHD1 mutants to restrict (green+) or not (red−) viral infection is indicated. b. The same samples as in A were separated on a 7% Phos-tag^TM SDS-PAGE gel. Arrowheads indicate that T592 phosphorylated SAMHD1 species which become undetectable upon T₅₉₂E mutation. Results of one representative experiment are shown (n = 3). c U937 cell lines (2–4 independently generated cell lines) stably expressing the indicated HA-SAMHD1 mutants were differentiated and next infected with the VSVg/HIV-1∆EnvEGFP virus and analyzed by flow cytometry 24 h later. The infection rate of parental U937 cells was set to 100. b Data (mean ± SD) from one representative experiment performed in three technical replicates are shown (n = 4 for U937, WT, T₅₉₂E, and T₅₉₂A; n = 2 for K₅₉₅A, T₅₉₂A_K₅₉₅R, T₅₉₂A_E₅₉₇Q, and T₅₉₂A_SIM2m). Statistical significance was assessed by one-way ANOVA test with Dunnett’s multiple comparison post test. ***p < 0.001, ****p < 0.0001. ns: not significant, p > 0.05. d A schematic representation of SAMHD1 variants harboring C-terminal deletions or expressed as SUMO1- or SUMO2-fusion proteins. The C-terminal diglycine motif of SUMO isoforms is changed into di-alanine to prevent conjugation (GG/AA). Residues of the CDK- and SUMO-consensus motif are underlined or bold and K595 and E597 are colored in red and blue, respectively. e U937 cell lines (≥2 independently generated cell lines) stably expressing the indicated HA-SAMHD1 mutants were analyzed as in (c). Statistical significance was assessed by one-way ANOVA test with Dunnett’s multi-comparison post test. ****p < 0.0001. ns not significant, p > 0.05. f The levels of dATP were quantified and normalized as in Fig. 3d. The dNTP levels of U937 were set to 100%. Bars show the mean ±  SD (n ≥ 2). Statistical significance was assessed by one-way ANOVA test with Dunnett’s multiple comparison post test. ****p < 0.0001. ns not significant, p > 0.05.