Abstract Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4 + T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood. We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion. In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled. Importance Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent anti-viral restriction factor, active against a broad range of DNA viruses and retroviruses. In myeloid and resting CD4 + T cells, SAMHD1 blocks reverse transcription of immunodeficiency virus 1 (HIV-1), not only inhibiting viral replication in these cell types, but also limiting the availability of reverse transcription products for innate sensing of HIV-1. Manipulating SAMHD1 activity could be an attractive approach to improve HIV-1 therapy or vaccination strategies. Anti-viral activity is strictly dependent on dephosphorylation of SAMHD1 residue T592, however the mechanistic consequence of T592 phosphorylation is still unclear. Here, we use BlaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated KI to study the influence of SAMHD1 T592 phosphorylation on anti-viral restriction and control of cellular dNTP levels in an endogenous context. By using this novel approach, we were able to genetically uncouple SAMHD1’s anti-viral and dNTPase activity with regard to regulation by T592 phosphorylation. This suggests that SAMHD1 dNTPase activity may not exclusively be responsible for the anti-lentiviral activity of SAMHD1 in myeloid cells. In addition, our toolkit may inspire further genetic analysis and investigation of SAMHD1-mediated restriction, as wells as its cellular function and regulation, leading to a deeper understanding of SAMHD1 and HIV-1 biology.

CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1