Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.

Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. 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 not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of additional SAMHD1-mediated anti-viral activity independent of T592 dephosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet a poor correlation between HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function, and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.IMPORTANCEWe introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.