Caitlin Shepard

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G1/G0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

To the Editor: Sterile alpha motif domain– and HD domain–containing protein 1 (SAMHD1) is a cellular dNTP triphosphohydrolase (dNTPase) that restricts HIV-1 replication in myeloid cells and resting CD4+ T cells by degrading dNTPs and limiting viral reverse transcription1–5. Purified recombinant SAMHD1 also has exonuclease activity when synthetic nucleic acids or HIV-1 gag and tat RNAs transcribed in vitro are used as substrates6. Ryoo et al.7 recently suggested that SAMHD1 restricts HIV-1 infection through its ribonuclease (RNase) activity by cleaving the viral RNA genome. By using SAMHD1 mutants purported to specifically retain dNTPase (SAMHD1Q548A) or RNase (SAMHD1D137N) activities, Ryoo et al.7,8 proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in nondividing cells. They also suggested that SAMHD1 phosphorylation at T592 negatively regulated its RNase activity7. To extend these findings7, we measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-dependent HIV-1 reverse transcription was bypassed. We co-transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phosphoablative, but dNTPase-active, mutant (SAMHD1T592A; refs. 9–11) into human embryonic kidney (HEK) 293T cells and assessed intracellular HIV-1 Gag protein synthesis and viral particle release in the supernatants. This transfection-based HIV-1 production is independent of reverse transcription requiring intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA–mediated gene expression. Intracellular HIV-1 Gag protein levels, p24 capsid levels in released HIV-1 virions and infectivity were not reduced by the ectopic expression of WT SAMHD1 or SAMHD1T592A mutant (Supplementary Fig. 1), which suggests that SAMHD1 cannot inhibit HIV-1 production after the reverse transcription step, regardless of its phosphorylation at T592. These findings are consistent with our previous results showing that the expression of WT SAMHD1 or of the SAMHD1T592A mutant in dividing cells does not restrict HIV-1 infection11,12. Our results suggest that SAMHD1 does not have broad nuclease activity, but do not rule out a specific nucleolytic interaction between SAMHD1 and incoming HIV-1 genomic RNA (gRNA). Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, we reproduced the key experiments of Ryoo et al.7. Their conclusion that SAMHD1 restricts HIV-1 through RNase activity was based on the differential activity of the SAMHD1 mutants, SAMHD1D137N and SAMHD1Q548A (ref. 7). We independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure that there were no other disabling mutations in the constructs. We then examined the HIV-1 restriction and intracellular dNTP regulation by these mutants and WT SAMHD1 by following the protocol of Ryoo et al.7. Our results show that both the SAMHD1D137N and SAMHD1Q548A mutants were expressed at similar levels to that of WT SAMHD1, and each efficiently restricted HIV-1 infection and decreased dATP, dGTP and dTTP levels in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (Fig. 1a–c). SAMHD1 expression did not significantly decrease dCTP levels, as compared to vector control cells (Fig. 1c, right), probably owing to the different biosynthesis pathway of dCTP. Notably, Ryoo et al.7 showed only dCTP levels, but not dATP, dGTP or dTTP levels, in SAMHD1expressing or control cells. Previous studies have used a SAMHD1D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation and HIV-1 restriction of SAMHD1 (refs. 13–15). The SAMHD1D137A mutant has no detectable dNTPase activity or HIV-1 restriction in vitro, owing to its inability to form a stable tetramer14,15. It is possible that the SAMHD1D137N mutant might be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. It is also possible that an in vitro dNTPase assay using purified recombinant SAMHD1 proteins might not fully reflect the dNTPase activity of SAMHD1 in PMA-differentiated U937 cells. However, these possibilities remain to be examined to explain how the SAMHD1D137N mutant restricts HIV-1 infection if its dNTPase activity is impaired. In contrast to the results of Ryoo et al.7, the SAMHD1D137N and SAMHD1Q548A mutants in our experiments do not have differing anti-HIV-1 activities, and neither lacks the ability to lower cellular dNTP levels. Ryoo et al.7 reported an approximately twofold decrease in HIV-1 gRNA levels in PMA-differentiated U937 cells expressing WT SAMHD1 or SAMHD1D137N, but not the SAMHD1Q548A mutant, as compared to the control cells at 3 h and 6 h postinfection (h.p.i.), which suggests SAMHD1-mediated HIV-1 gRNA degradation7. By contrast, we detected comparable levels of HIV-1 gRNA in PMAdifferentiated U937 cells expressing SAMHD1 (WT, SAMHD1D137N and SAMHD1Q548A mutants) and the vector control cells at 1, 3 and 6 h.p.i., respectively (Fig. 1d), showing that, in our study, SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 h.p.i, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that the expression of WT SAMHD1, SAMHD1D137N and SAMHD1Q548A mutants significantly reduced HIV-1 late reverse transcription products as compared to the vector control cells (Fig. 1e), correlating well with a reduced intracellular dNTP pool (Fig. 1c). Thus, in our study, these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction. Seamon et al.16 reported that trace exonuclease activities of recombiSAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity

SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.

Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect

SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.

An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease.

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti–influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg2+ or Mn2+ ions, which are critical for its catalytic function. There is great interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We focused here on a series of β-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, in order to build a three-dimensional pharmacophore model. In addition, we developed a molecular beacon (MB)–based PA-Nter assay that enabled us to compare the inhibition of Mn2+ versus Mg2+, the latter probably being the biologically relevant cofactor. This real-time MB assay allowed us to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC50 values comparable to that of the prototype L-742,001 (i.e., below 2 μM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg2+ or Mn2+. Three compounds (10, with a pyrrole scaffold, and 40 and 41, with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values 64–95 μM) and were proven to affect viral RNA synthesis. Our approach of integrating complementary enzymatic, cellular, and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.

Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection

Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.

HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.

A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse

The triphosphohydrolase SAMHD1 (sterile α motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro. dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication.

The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

BackgroundThe restriction factor SAMHD1 regulates intracellular nucleotide level by degrading dNTPs and blocks the replication of retroviruses and DNA viruses in non-cycling cells, like macrophages or dendritic cells. In patients, inactivating mutations in samhd1 are associated with the autoimmune disease Aicardi-Goutières Syndrome (AGS). The accumulation of intracellular nucleic acids derived from endogenous retroelements thriving in the absence of SAMHD1 has been discussed as potential trigger of the autoimmune reaction. In vitro, SAMHD1 has been found to restrict endogenous retroelements, like LINE-1 elements (L1). The mechanism, however, by which SAMHD1 blocks endogenous retroelements, is still unclear.ResultsHere, we show that SAMHD1 inhibits the replication of L1 and other endogenous retroelements in cycling cells. By applying GFP- and neomycin-based reporter assays we found that the anti-L1 activity of SAMHD1 is regulated by phosphorylation at threonine 592 (T592). Similar to the block of HIV, the cofactor binding site and the enzymatic active HD domain of SAMHD1 proofed to be essential for restriction of L1 elements. However, phosphorylation at T592 did not correlate with the dNTP hydrolase activity of SAMHD1 in cycling 293T cells suggesting an alternative mechanism of regulation. Interestingly, we found that SAMHD1 binds to ORF2 protein of L1 and that this interaction is regulated by T592 phosphorylation. Together with the finding that the block is also active in cycling cells, our results suggest that the SAMHD1-mediated inhibition of L1 is similar but not identical to HIV restriction.ConclusionOur findings show conclusively that SAMHD1 restricts the replication of endogenous retroelements in vitro. The results suggest that SAMHD1 is important for maintaining genome integrity and support the idea of an enhanced replication of endogenous retroelements in the absence of SAMHD1 in vivo, potentially triggering autoimmune diseases like AGS. Our analysis also contributes to the better understanding of the activities of SAMHD1 in antiviral defense and nucleotide metabolism. The finding that the phosphorylation of SAMHD1 at T592 regulates its activity against retroelements but not necessarily intracellular dNTP level suggests that the dNTP hydrolase activity might not be the only function of SAMHD1 important for its antiviral activity and for controlling autoimmunity.