The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

SAMHD1 restricts human immunodeficiency virus-1 (HIV-1) infection of dendritic and other myeloid cells at an early stage in the replication cycle. SIVsm/HIV-2 lineage viruses counteract SAMHD1-mediated restriction by encoding Vpx, a virion-packaged accessory protein that targets SAMHD1 for degradation. We show that SAMHD1 restricts HIV-1 infection of monocyte-derived macrophages (MDM) by hydrolyzing the cellular deoxynucleotide triphosphates (dNTP), reducing their level to below that required for the synthesis of the viral genomic DNA. Vpx prevented the SAMHD1-mediated decrease in dNTP. The restriction was partially alleviated in MDM by the addition of exogenous deoxynucleosides. HIV-1 with a V148I mutation in reverse transcriptase that lowers its affinity for dNTP was particularly sensitive to SAMHD1-mediated restriction. Nucleotide starvation could serve as a mechanism to protect cells from infection by a wide variety of infectious agents that replicate through a DNA intermediate.

SAMHD1 restricts HIV-1 replication in myeloid and quiescent CD4(+) T cells. Here, we show that SAMHD1 restriction activity is regulated by phosphorylation. SAMHD1 interacts with cyclin A2/cdk1 only in cycling cells. Cyclin A2/CDK1 phosphorylates SAMHD1 at the Threonine 592 residue both in vitro and in vivo. Phosphorylation of SAMHD1 Thr592 correlates with loss of its ability to restrict HIV-1. Indeed, while PMA treatment of proliferating THP1 cells results in reduced Thr592 phosphorylation, activation of resting peripheral blood mononuclear cells (PBMCs) and purified quiescent CD4(+) T cells results in increased phosphorylation of SAMHD1 Thr592. Interestingly, we found that treatment of cells by type 1 interferon reduced Thr592 phosphorylation, reinforcing the link between the phosphorylation of SAMHD1 and its antiviral activity. Unlike wild-type SAMHD1, a phosphorylation-defective mutant was able to restrict HIV-1 replication in both PMA-treated and untreated cells. Our results uncover the phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 as a key regulatory mechanism of its antiviral activity.