Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect

Abstract

SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…It is still controversial if phosphorylation at T592 impairs SAMHD1 dNTPase activity, since in vitro data obtained with purified SAMHD1 variants do not match the effects of the same mutants on the dNTP pools of transfected cells [15,20–22]. Considering that overexpression of an ectopic protein per se might alter the physiological conditions, we chose to investigate the phosphorylation of the endogenous protein.…”

“…In live cells, the effects of SAMHD1 phosphorylation were investigated by ectopic over-expression of SAMHD1 mutants and the restriction of viral infection or dNTP pool decrease, both readouts of SAMHD1 activity. In PMA differentiated U937 cells, phosphomimetic SAMHD1 mutants lacked retroviral restriction although they decreased cellular dNTP concentrations as did wild type SAMHD1 and its non-phosphorylatable mutants [15,20–22]. In proliferating cells, none of the tested SAMHD1 variants blocked retroviral infection, presumably due to the high expression of RNR that opposed the catabolic activity of SAMHD1[22].…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…Some evidence suggests phosphorylation negatively modulates SAMHD1 tetramerization and dNTPase activity (81, 99, 127), and this diminished dNTPase capacity is responsible for increased intracellular dNTP pools (93, 120, 121, 128). Other studies find limited or no effect of phosphorylation of SAMHD1 on catalytic activity(81, 108, 116, 117, 129), oligomerization equilibrium and allosteric activation(77, 129), or nucleic acid binding (83). Complicating the model further, phosphorylation may affect SAMHD1 substrate specificity (78).…”

“…Studies attempting to shed light on these discrepancies reveal that P-T592 results in altered kinetics of tetramer association and dissociation and expedited regulatory nucleotide release (77, 129). P-T592 is also less likely to form the activated tetramer at low concentration of activating nucleotides (78, 81).…”

“…Evidence exists to suggest that tetramerization deficient SAMHD1 enzymes may still be able to exert antiviral effects (129, 170). Additionally, the antiviral capacity of SAMHD1 is strictly correlated with its phosphorylation status on its C-terminus at T592, even while debate as to the actual effect of P-T592 on SAMHD1 catalytic activity persists.…”