SUMMARY SAMHD1 is a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and inhibits the ability of retroviruses, notably HIV-1, to infect myeloid cells. Although SAMHD1 is expressed in both cycling and noncycling cells, the antiviral activity of SAMHD1 is limited to noncycling cells. We determined that SAMHD1 is phosphorylated on residue T592 in cycling cells but that this phosphorylation is lost when cells are in a noncycling state. Reverse genetic experiments revealed that SAMHD1 phosphorylated on residue T592 is unable to block retroviral infection, but this modification does not affect the ability of SAMHD1 to decrease cellular dNTP levels. SAMHD1 contains a target motif for cyclin-dependent kinase 1 (cdk1) (592TPQK595), and cdk1 activity is required for SAMHD1 phosphorylation. Collectively, these findings indicate that phosphorylation modulates the ability of SAMHD1 to block retroviral infection without affecting its ability to decrease cellular dNTP levels.

The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to block HIV-1 and SIVmac infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine–aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIVmac. We demonstrated that the HD domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIVmac. These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV).

Highlights d The susceptibility of CD4 + T cell subsets to HIV-1 matches their metabolic activity d HIV-1 selectively infects CD4 + T cells with enhanced glycolysis and OXPHOS d Inhibition of metabolic activities blocks HIV-1 replication d Suboptimal inhibition of glycolysis impairs amplification of HIV-1 reservoirs

The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration.

BackgroundSAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation.ResultsHere, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1.ConclusionsWe mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.