Figure 3: Restriction of HBV by SAMHD1 is dependent upon dNTPase activity, is inhibited by phosphorylation of T592 and is rescued by addition of deoxynucleosides.

(a,b) HepG2.2.15 cells were transfected with 1 μg of plasmids coding for wild-type or mutant FLAG-tagged SAMHD1 or with 1 μg of empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements for 48 hours. (a) Detection of overexpressed FLAG-SAMHD1 by Western blotting. Detection of actin was used as a loading control. (b) HBV DNA from the supernatant was quantified by qPCR. The mean ± SEM of technical triplicates from one representative experiment out of three independent experiments is represented. (c) HepG2 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements to arrest the cell cycle. When indicated, 10 mM of hydroxyurea (HU) were added at the time of medium change. Quantification of dATP levels 3 days post medium change were determined by single-nucleotide incorporation assay. The mean ± SEM of technical duplicates is represented. (d) HepG2.2.15 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or with an empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements. When indicated, deoxynucleosides (dNs, 2 mM each) or solvent were added at the time of medium change. HBV DNA from the supernatant was quantified by qPCR 3 days post medium change. For each independent experiment, fold-changes to empty vector were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is represented. (b,d) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (**p < 0.005; ***p < 0.0005; ****p < 0.00005; n.s.: non-significant).