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.
Highlights
Human SAMHD1 protein is an innate immunity factor involved in the regulation of interferon signaling and retroviral restriction
There are clear deviations from the vertical in the appearance of the van Holde-Weischet (vHW) plots for the higher molecular weight species, which is indicative of the graduate tetramer dissociation occurring during the run as dGTPαSis depleted by the dNTPase activity of SAMHD1
We do not observe any significant effects of the T592D mutation on the ability of SAMHD1 to tetramerize in the presence of dATP and GTP when we measure the amount of SAMHD1 tetramer as a function of nucleotide concentration
Summary
Human SAMHD1 protein is an innate immunity factor involved in the regulation of interferon signaling and retroviral restriction. Phosphomimetic mutants of SAMHD1, T592D and T592E, display a puzzling phenotype: they appear to be intact in their ability to deplete cellular dNTP pools, but are unable to restrict HIV infection[15,16,17,18]. It was suggested that the tetramer is the active form of the enzyme and that the dependence of tetramerization on dNTP binding is essential for controlling cellular dNTP concentrations This regulatory mechanism bears some similarity to regulation of ribonucleotide reductase and thymidine kinase, two key enzymes in deoxynucleotide metabolism which are both tetrameric and allosterically controlled by nucleotide binding[33,34,35]
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