Abstract
SUMMARYSAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of noncycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.
Highlights
SAMHD1 is a member of the HD domain family of enzymes, and the protein displays nucleotide-dependent dinucleotide triphosphate triphophohydrolase activity previously described for other members of this protein family (Lahouassa et al, 2012; Powell et al, 2011; Goldstone et al, 2011; Beauchamp and Richardson, 1988; Kornberg et al, 1958; Seto et al, 1988)
The dinucleotide triphosphate triphophohydrolase (dNTPase) activity of SAMHD1 is thought to be central for its antiretroviral function, because SAMHD1 blocks retroviral replication before completion of reverse transcription (Fujita et al, 2008; Goujon et al, 2007; Laguette et al, 2011) and the SAMHD1-mediated decrease in the dinucleotide triphosphate levels in myeloid cells correlates with the inability of lentiviruses to undergo reverse transcription (Kim et al, 2012; Lahouassa et al, 2012)
We observed that addition of 5 mM DTT or 5 mM reduced glutathione to the reaction buffer dramatically increased dNTPase activity of SAMHD1 (Figure S1A), whereas brief incubation with hydrogen peroxide inhibited the enzyme (Figure S1B)
Summary
Discoveries that SAMHD1 mutations cause Aicardi-Goutieres syndrome (AGS) (Rice et al, 2009) and that SAMHD1 restricts HIV-1 replication in non-cycling immune cells (Baldauf et al, 2012; Berger et al, 2011; Descours et al, 2012; Hrecka et al, 2011; Laguette et al, 2011) established the role of SAMHD1 as an innate immunity factor involved in interferon signaling and retroviral restriction.SAMHD1 is a member of the HD domain family of enzymes, and the protein displays nucleotide-dependent dinucleotide triphosphate triphophohydrolase (dNTPase) activity previously described for other members of this protein family (Lahouassa et al, 2012; Powell et al, 2011; Goldstone et al., 2011; Beauchamp and Richardson, 1988; Kornberg et al, 1958; Seto et al, 1988). Several tetramerization-defective mutants of SAMHD1 that display a striking dNTPase defect in vitro are intact in their ability to deplete dNTPs in immune cells and to restrict retroviral replication (Bhattacharya et al, 2016; Brandariz-Nunez et al, 2013). Another poorly understood aspect of SAMHD1 function involves protein phosphorylation on threonine 592 (T592). Phosphomimetic mutations of T592 abolish restriction but do not affect the dNTPase activity of the protein in vitro or its ability to deplete dNTPs in cells (Bhattacharya et al, 2016; Welbourn et al, 2013; White et al, 2013b), raising additional questions about the relationship between dNTPase activity
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