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 HIV-1 restriction factor SAMHD11,2 is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool3-5. However, the phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels6-8, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1, we identify SAMHD1 mutants that are RNase-positive but dNTPase-negative (SAMHD1D137N) or RNase-negative but dNTPase-positive (SAMHD1Q548A). The allosteric mutant SAMHD1D137N is able to restrict HIV-1 infection, whereas the AGS mutant SAMHD1Q548A is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, the phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in vivo and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.

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).

BackgroundThe IFN-α-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Genetic evidence suggested that capsid is the viral determinant for restriction by MxB. This work explores the ability of MxB to bind to the HIV-1 core, and the role of capsid-binding in restriction.ResultsWe showed that MxB binds to the HIV-1 core and that this interaction leads to inhibition of the uncoating process of HIV-1. These results identify MxB as an endogenously expressed protein with the ability to inhibit HIV-1 uncoating. In addition, we found that a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to capsid. The use of this small-molecule identified the MxB binding region on the surface of the HIV-1 core. Domain mapping experiments revealed the following requirements for restriction: 1) MxB binding to the HIV-1 capsid, which requires the 20 N-terminal amino acids, and 2) oligomerization of MxB, which is mediated by the C-terminal domain provides the avidity for the interaction of MxB with the HIV-1 core.ConclusionsOverall our work establishes that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1. Moreover, we demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0068-x) contains supplementary material, which is available to authorized users.

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.

BackgroundExpression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration.ResultsThis work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection.ConclusionsThese results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.

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bThe stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects on HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure the stability of in vitro-assembled HIV-1 CA-NC complexes. The assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core. Interestingly, stabilization of in vitro-assembled HIV-1 CA-NC complexes is not due solely to macromolecular crowding, suggesting the presence of specific cellular factors that stabilize the HIV-1 core. By using our novel assay, we measured the abilities of different drugs, such as PF74, CAP-1, IXN-053, cyclosporine, Bi2 (also known as BI-2), and the peptide CAI, to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes. Interestingly, we found that PF74 and Bi2 strongly stabilized HIV-1 CA-NC complexes. On the other hand, the peptide CAI destabilized HIV-1 CA-NC complexes. We also found that purified cyclophilin A destabilizes in vitro-assembled HIV-1 CA-NC complexes in the presence of cellular extracts in a cyclosporine-sensitive manner. In agreement with previous observations using the fate-of-the-capsid assay, we also demonstrated the ability of recombinant CPSF6 to stabilize HIV-1 CA-NC complexes. Overall, our findings suggested that cellular extracts specifically stabilize the HIV-1 core. We believe that our assay can be a powerful tool to assess HIV-1 core stability in vitro.