SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.
Author Summary SAMHD1 is a restriction factor that blocks infection of certain immune cells by HIV-1. It was discovered to be an enzyme that catalyses the breakdown of dNTPs, suggesting that it inhibits HIV-1 replication by reducing cellular dNTP pools to such low levels that reverse transcriptase cannot function. However, recently, alternative mechanisms have been proposed. SAMHD1 is also regulated by phosphorylation, although the effects of phosphorylation on protein function are unclear. In order to address these issues, we carried out combined structural and virological studies and have demonstrated that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Disrupting the tetramer in various ways always abolished restriction but had differing effects on enzyme activity in vitro. SAMHD1 phosphorylation also prevented restriction and tetramer formation but without affecting enzyme catalysis under steady-state dNTP conditions. However phosphorylated SAMHD1 was unable to catalyse dNTP turnover at very low nucleotide levels that more accurately represent conditions in the cells in which restriction takes place. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity and substantiate that degradation of dNTPs by SAMHD1 is sufficient to restrict HIV-1.