Fig. 4

Phosphorylation at threonine 603 regulates the antiviral activity of murine SAMHD1. a THP-1 KO cells expressing murine SAMHD1 isoform 1, isoform 1 T603A, isoform 1 T603D, isoform 2, or control cells (luc) were incubated with (+PMA) or without PMA (−PMA) for 16 h, lysed and analyzed by SDS-PAGE and immunoblot. Membranes were probed with anti-HSP90, anti-SAMHD1 MAb 3F5, and anti-pSAMHD1-T592 antibody to determine SAMHD1 phosphorylation. b THP-1 KO cells expressing mouse isoform 1, isoform 1 T603A, isoform 1 T603D, isoform 2, or control cells (luc) were differentiated with 50 nM PMA for 24 h or left untreated. After 24 h, samples were normalized to cell number, lysed and dNTP level of 1 × 10⁶ cells were determined using liquid chromatography tandem mass spectrometry. The intracellular concentration of dNTPs (nM) in cycling and noncycling cells is depicted. The dNTP levels are displayed as the average of three measurements with error bars indicating the standard deviation. c THP-1 SAMHD1 knockout cells (THP KO) stably expressing mouse iso1, T603A, T603D, iso2 or firefly luciferase expressing control cells (luc) were left untreated (−PMA) or differentiated with 50 nM PMA (+PMA) for 24 h prior to infection with VSV-G/HIV-GFP at a MOI of 1. Three days postinfection, cells were lysed and viral infectivity was quantified by flow cytometry. Viral infectivity was normalized on control cell infection (luc). Error bars indicate the standard deviation of triplicate infections. One out of three independent experiments is shown