SAM Domain And HD Domain-containing Protein 1 Research Articles

SAMHD1 … and Viral Ways around It.

The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.

Expression of the novel tumour suppressor sterile alpha motif and HD domain‐containing protein 1 is an independent adverse prognostic factor in classical Hodgkin lymphoma

The expression patterns and prognostic significance of sterile alpha motif and HD domain-containing protein 1 (SAMHD1) protein in the neoplastic Hodgkin and Reed Sternberg (HRS) cells of Hodgkin lymphoma (HL) were investigated in a cohort of 154 patients with HL treated with standard regimens. SAMHD1 expression was assessed by immunohistochemistry using diagnostic lymph node biopsies obtained prior to treatment. Using an arbitrary 20% cut-off, SAMHD1 was positive in HRS cells of 48/154 (31·2%) patients. SAMHD1 expression was not associated with clinicopathologic parameters, such as age, gender, stage or histologic subtype. In 125 patients with a median follow-up of 90months (7-401months), SAMHD1 expression in HRS cells significantly correlated with inferior freedom from progression (FFP) (P=0·025), disease-specific survival (DSS) (P=0·013) and overall survival (OS) (P=0·01). Importantly, in multivariate models together with disease stage, histology subtype and type of treatment as covariates, SAMHD1 expression retained an independent significant association with unfavourable FFP (P=0·005) as well as DSS (P=0·022) and OS (P=0·018). These findings uncover the significance of a novel, adverse prognostic factor in HL that may have therapeutic implications since SAMHD1 inhibitors are now available for clinical use.

Repurposing Nelarabine to Induce Differentiation of Acute Myeloid Leukemia

The success of all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL) pioneered the concept of differentiation therapy. However, comparable approaches to overcome differentiation blockage for non-APL acute myeloid leukemia (AML) are hampered by lack of an effective drug discovery platform. Recently, we analyzed gene signatures of compounds (ATRA, arsenic trioxide, zalcitabine, and sodium butyrate) that trigger myeloid differentiation in the NCI-60 collection datasets and identified CD38 as the top gene upregulated by differentiation induction. We next initiated an in silico screen in the DTP database of >20,000 compounds to identify compounds that increase CD38 levels. Among those retrieved from "CellMiner" with CD38 as input, we assessed the top 193 available from NCI (r>0.6, p=0) for effects on differentiation utilizing a conditional murine myeloid differentiation-arrest model overexpressing estrogen receptor-HoxA9 (ER-HoxA9) fusion proteins (Cell, 2016). We identified NSC755985 (Nelarabine, NEL) in that screen. NEL is an orphan drug approved to treat relapsed or refractory T-cell acute lymphoblastic leukemia (ALL). NEL at clinically achievable doses (Cmax: 6.73 μM~ 26.91 μM, at a proposed adult dosing schedule of 1,500 mg/m2/day) markedly induced primary AML cell differentiation and death while sparing normal hematopoietic cells (AML vs normal, IC50: 14.7 ± 4.3 μM, n=7, vs 45.3 ± 1.3 μM, n=3), suggesting a therapeutic window in AML. Ex vivo NEL treatment compromised BM engraftment of CD34+ cells from one primary AML specimen in immunodeficient NSG mice at 12 weeks post-transplant (human CD45+ cells in BM: NEL 0.73% vs vehicle 33.42%, n=6/group, p<0.01). NEL administration in vivo (130 mg/kg/day, i.v. for 5 consecutive days) reduced leukemic burden of NSG mice xenografted with luciferase-expressing U937 cells (Radiance: NEL 2.83×107 vs vehicle 1.65×108 photons/s/cm2, n=9/group,p<0.01) and extended mouse survival. Transcriptome analyses in U397 cells and primary AML specimens revealed that NEL treatment upregulated RAS-related pathways. NEL-elicited RAS activation was confirmed by pull-down assay using a GST-Raf1-RBD affinity probe, followed by blotting with a pan-RAS antibody. We performed functional analysis by infecting ER-HoxA9 cells with lentiviral vector expressing oncogenic RAS and observed enhanced myeloid differentiation, as evidenced by increased CD11b/GFP levels relative to MOCK-infected controls. Given that NEL's active metabolite Ara-GTP perturbs guanine nucleotide metabolism, we asked if NEL-evoked RAS activation was associated with accrual of intracellular GTP. HPLC/MS analyses of U937 cells showed that NEL treatment resulted in a marked increase in GTP (approximately 5-fold higher than baseline at sub-millimolar levels) which was secondary to Ara-GTP. Importantly, either electroporation of GTP into U937 cells or indirect introduction of GTP by addition of guanine utilizing purine salvage pathways activated RAS and recapitulated differentiation induction phenotypes. To determine whether AML cells with higher RAS activity exhibited greater NEL sensitivity, we pretreated U937 cells with a RAS agonist KRA-553 or ectopically expressed RAS mutants and observed enhanced NEL inhibitory effects in both cases. We also observed enhanced vulnerability to NEL treatment in MLL-AF9 transformed murine hematopoietic cells from KrasLox-Stop-Lox (LSL) G12D/+/Vav-Cre mice (Blood, 2009) versus Cre+ counterparts. Relevant to AML line THP-1 which is poorly responsive to NEL (IC50>100 µM), we observed extremely low levels of Ara-GTP, no GTP increase or RAS hyperactivation after NEL treatment; Ara-GTP is inactivated by SAM domain and HD domain-containing protein 1 (SAMHD1), a dNTP hydrolase, whose high expression reportedly underlies NEL resistance in T-ALL. Indeed, SAMHD1 deletion remarkably increased RAS activity in THP-1 cells treated with NEL, thereby fully reversing NEL resistance. Our study provides a preclinical basis for testing NEL efficacy in a large cohort of AML patients, given that RAS activity is generally high in AML, or even against other malignancies harboring RAS mutations, which are considered "undruggable". Additionally, further study to test whether SAMHD1 inhibition enhances NEL efficacy against RAS active cancers is warranted. Disclosures Marcucci: Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Novartis: Speakers Bureau; Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Merck: Other: Research Support (Investigation Initiated Clinical Trial); Abbvie: Speakers Bureau. Sykes:Clear Creek Bio: Current equity holder in private company, Other: co-founder.

Downregulation of GLI3 Expression Mediates Chemotherapy Resistance in Acute Myeloid Leukemia.

Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.

The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-β, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-β mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

281P - Selective depletion of tumour-associated SAMHD1 by HSP90 inhibitors enhances the anti-AML effect of cytarabine

BackgroundAcute myeloid leukemia (AML) is the most common acute type of leukemia in adults, with a disappointing prognosis and high death rate. Cytarabine (ara-C) is a primary first-line chemotherapy drug used in the treatment of AML, and resistance to it represents a major source of treatment failure. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), the only known human dNTPase, is known to attenuate ara-C cytotoxicity in AML cells, resulting in treatment failure and a poor prognosis. Therefore, selective inhibition of SAMHD1 in AML should prove a promising strategy for increasing the efficacy of ara-C. Nevertheless, no effective strategy to target SAMHD1 is available yet. MethodsCell culture, Monocyte isolation and differentiation, Cytotoxicity assays, Protein extraction and Western blot analysis, Immunoprecipitation, Immunofluorescent staining, Tissue staining, Mass spectrum system, mRNA analysis, RNA silencing, Flow cytometry, Orthotopic AML animal model, Heterotopic AML animal model. ResultsHere, we report that SAMHD1 is a novel HSP90-dependent protein in various tumor types. Pharmacological inhibition of HSP90 led to SAMHD1 depletion in a wide range of tumor cell types but had no or minimally effect on SAMHD1 of peripheral blood mononuclear cells (PBMCs) or macrophages from healthy donors due to the phosphorylation status on Thr 592. The combination of ara-C and HSP90 inhibitors showed a synergistic effect in killing AML cells. The HSP90 inhibitor also significantly enhanced the therapeutic efficacy of ara-C in both orthotopic and heterotopic models, alleviating tumor burden and prolonging survival. ConclusionsOverall, these data suggest that the addition of an HSP90 inhibitor to ara-C-based chemotherapy represents a potential new treatment strategy for AML patients. Legal entity responsible for the studyXiaofang Yu. FundingNational Natural Science Foundation of China (81772169). DisclosureAll authors have declared no conflicts of interest.

Effect of induced dNTP pool imbalance on HIV-1 reverse transcription in macrophages

BackgroundTerminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages.ResultsThe transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20–30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment.ConclusionCollectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.

Expression and Relationship of SAMHD1 with Other Apoptotic and Autophagic Genes in Acute Myeloid Leukemia Patients

Background: SAM domain- and HD domain-containing protein 1 (SAMHD1) is a cellular enzyme which is responsible for blocking replication in viruses and participates in the progression of many cancers. Objective: The aim of this study was to correlate the expression level of SAMHD1 with other apoptotic and autophagic genes in acute myeloid leukemia (AML) patients. Methods: In the present study, mRNA levels of SAMHD1 with other apoptotic and autophagic-related genes were evaluated in patients who were newly diagnosed with AML. Results: SAMHD1, Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in the AML group compared to the non-AML group (p < 0.05). SAMHD1 expression did not correlate with the other genes, while most apoptotic genes were positively correlated with each other. SAMHD1 expression was not associated with the blood routine or blast percentage of the AML patients, while Bax, Bak, cIAP2, and LC3 were significantly correlated with white blood cells. No statistically significant differences were found between the studied genes and prognosis stratifications, but Bcl-xl, Bak, cIAP1, and Mcl-1, LC3 were expressed at lower levels in the unfavorable AML group compared to the controls. Conclusion: SAMHD1 and Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in AML patients, while there were no significant differences in the clinical characteristics and prognosis with reference to SAMHD1 expression.

Abstract 1744: SAMHD1 expression and impact on clinical outcome in diffuse large B-cell lymphoma: a potential therapeutic target

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. While standard treatment with rituximab, cyclophosphamide, doxorubicin, vincris- tine, and prednisone (R-CHOP) can cure DLBCL, failed patients have poor outcome. Thus, a search for stand-alone or adjuvant therapy to improve patient’s outcome is important. Sam and HD domain-containing protein 1 (SAMHD1) is a well-established deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase. Its dNTPase activity has been associated with cellular defence mechanism against infection by depleting cellular dNTP pool bellow level required for viral replication. SAMHD1 mutations have been associated with Aicardi Goutières (AGS) and cancers such as chronic lymphocytic leukemia (CLL), demonstrating its relevance beyond viral infection. In addition, it has been shown that SAMHD1 expression in Recently, our lab has defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR). SAMHD1 expression and its diagnostic or therapeutic relevance in DLBCL is not known. Thus, we analyzed SAMHD1 expression in patients with DLBCL and determined its correlation with clinical outcome. In addition, we studied the SAMHD1 expression in germinal center B cell (GCB) and activated B cell (ABC) DLBCL cells lines. Also, we examined the possibility of depleting SAMHD1 using a virus like particles (VLPs) containing viral protein x (Vpx) in DLBCL cell lines. Finally, we determined the effect on cells sensitivity to standard DLBCL therapy, R-CHOP, and other DNA damage inducing agents. Low SAMHD1 expression correlates with improved overall survival. SAMHD1 was downregulated in ABC as compared to GCB DLBCL cell lines and SAMHD1 depletion sensitizes DLBCL cell lines to R-CHOP and other DNA damage inducing agents. These findings suggest that SAMHD1 is a potential therapeutic target to improve the DLBCL patient’s clinical outcome. Citation Format: Waaqo Daddacha. SAMHD1 expression and impact on clinical outcome in diffuse large B-cell lymphoma: a potential therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1744.

Abstract 3497: SAMHD1 is a Novel Biomarker and Therapeutic Target for Radiation Therapy and PARP Inhibition in Breast Cancer

Abstract SAMHD1 is a Novel Biomarker and Therapeutic Target for Radiation Therapy and PARP Inhibition in Breast Cancer Ashley Schlafstein, Waaqo Daddacha, David S. Yu Background/Introduction: Patients with breast cancer are often treated by ionizing radiation (IR) and PARP inhibitors; however, the effectiveness of these treatments is often limited by lack of response and/or resistance. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxyribonucleotide triphosphate (dNTP) triphosphohydrolase with a well-established role in HIV-1 restriction by depleting dNTPs required for reverse transcription and replication. SAMHD1 is also dysregulated in breast and other cancers and in autoimmune disease. In addition to its well-established dNTPase activity, our lab has defined a novel role for SAMHD1 in DNA end resection to facilitate DNA double strand break (DSB) repair by homologous recombination (HR). Because of its role in DNA DSB repair and its link to breast cancer, we hypothesized that SAMHD1 could be used as a novel biomarker and target for breast cancer therapy. Methods: We queried the TCGA database for patients with breast cancer who were treated with or without IR to determine if there was an association of SAMHD1 expression level with clinical outcome. We also determined if targeting SAMHD1 for proteasomal degradation with novel virus like particles (VLPs) containing Vpx, a viral accessory protein, can sensitize breast cancer cells and tumors to DNA damaging agents. Results: In patients with breast cancer treated with IR, low SAMHD1 expression was associated with a statistically significant improvement in overall survival, while for those not treated with IR, no significant difference in association of SAMHD1 expression with outcome was observed. Breast cancer cells and tumors treated with VLPs containing Vpx were hypersensitive to IR and PARP inhibitor. Conclusion: Our results identify SAMHD1 as a novel biomarker for stratifying breast cancer patients for treatment with IR. In addition, targeting SAMHD1 with VLPs containing Vpx may be a novel therapeutic strategy for sensitizing breast cancer to IR, PARP inhibitors, and other agents that induce DNA DSBs. Citation Format: Elizabeth Thompson. SAMHD1 is a Novel Biomarker and Therapeutic Target for Radiation Therapy and PARP Inhibition in Breast Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3497.

Kinetics of Early Innate Immune Activation during HIV-1 Infection of Humanized Mice.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with aberrant immune activation; however, most model systems for HIV-1 have been used during established infection. Here, we utilize ultrasensitive HIV-1 quantification to delineate early events during the eclipse, burst, and chronic phases of HIV-1 infection in humanized mice. We show that very early in infection, HIV-1 suppresses peripheral type I interferon (IFN) and interferon-stimulated gene (ISG) responses, including the HIV-1 restriction factor IFI44. At the peak of innate immune activation, prior to CD4 T cell loss, HIV-1 infection differentially affects peripheral and lymphoid Toll-like receptor (TLR) expression profiles in T cells and macrophages. This results in a trend toward an altered activation of nuclear factor κB (NF-κB), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3). The subsequent type I and III IFN responses result in preferential induction of peripheral ISG responses. Following this initial innate immune activation, peripheral expression of the HIV-1 restriction factor SAM domain- and HD domain-containing protein 1 (SAMHD1) returns to levels below those observed in uninfected mice, suggesting that HIV-1 interferes with their basal expression. However, peripheral cells still retain their responsiveness to exogenous type I IFN, whereas splenic cells show a reduction in select ISGs in response to IFN. This demonstrates the highly dynamic nature of very early HIV-1 infection and suggests that blocks to the induction of HIV-1 restriction factors contribute to the establishment of viral persistence.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection is restricted to humans and some nonhuman primates (e.g., chimpanzee and gorilla). Alternative model systems based on simian immunodeficiency virus (SIV) infection of macaques are available but do not recapitulate all aspects of HIV-1 infection and disease. Humanized mice, which contain a human immune system, can be used to study HIV-1, but only limited information on early events and immune responses is available to date. Here, we describe very early immune responses to HIV-1 and demonstrate a suppression of cell-intrinsic innate immunity. Furthermore, we show that HIV-1 infection interacts differently with innate immune responses in blood and lymphoid organs.

Multiple Autoimmune Disorders in Aicardi-Goutières Syndrome

BackgroundAicardi-Goutières syndrome is an early-onset encephalopathy with presumed immune pathogenesis caused by inherited defects in nucleic acid metabolism. It is a model disease to study systemic autoimmunity, and there are many clinical, genetic, and basic science considerations that underline a possible overlap between Aicardi-Goutières syndrome and systemic lupus erythematosus. ResultsWe describe a 15-year-old girl with Aicardi-Goutières syndrome due to compound heterozygous pathogenic variants in SAMHD1 (sterile alpha motif domain and HD domain-containing protein 1). Over time, she developed multiple autoimmune diseases (vitiligo, alopecia areata, immune thrombocytopenia, positive antithyroglobulin antibodies) without positive antinuclear antibody or features of systemic lupus erythematosus. Her thrombocytopenia was refractory to treatment with corticosteroids and intravenous immunoglobulin but responded to a standard course of rituximab. ConclusionThis is the first report of a multiple autoimmune syndrome in a patient with molecularly proven Aicardi-Goutières syndrome. This study illustrates an emerging pattern of the natural history of Aicardi-Goutières syndrome characterized by early encephalopathic presentation followed by symptoms of systemic autoimmunity.

IL-15 regulates susceptibility of CD4+ T cells to HIV infection.

HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4+ memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the HIV reservoir established. Therefore, we investigated the molecular and cellular impact of IL-15 on CD4+ T-cell infection. We found that IL-15 stimulation induces SAM domain and HD domain-containing protein 1 (SAMHD1) phosphorylation due to cell cycle entry, relieving an early block to infection. Perturbation of the pathways downstream of IL-15 receptor (IL-15R) indicated that SAMHD1 phosphorylation after IL-15 stimulation is JAK dependent. Treating CD4+ T cells with Ruxolitinib, an inhibitor of JAK1 and JAK2, effectively blocked IL-15-induced SAMHD1 phosphorylation and protected CD4+ T cells from HIV infection. Using high-resolution single-cell immune profiling using mass cytometry by TOF (CyTOF), we found that IL-15 stimulation altered the composition of CD4+ T-cell memory populations by increasing proliferation of memory CD4+ T cells, including CD4+ T memory stem cells (TSCM). IL-15-stimulated CD4+ TSCM, harboring phosphorylated SAMHD1, were preferentially infected. We propose that IL-15 plays a pivotal role in creating a self-renewing, persistent HIV reservoir by facilitating infection of CD4+ T cells with stem cell-like properties. Time-limited interventions with JAK1 inhibitors, such as Ruxolitinib, should prevent the inactivation of the endogenous restriction factor SAMHD1 and protect this long-lived CD4+ T-memory cell population from HIV infection.

Reversible Human Immunodeficiency Virus Type-1 Latency in Primary Human Monocyte-Derived Macrophages Induced by Sustained M1 Polarization

We have reported that short-term stimulation of primary human monocyte-derived macrophages (MDM) with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), i.e. M1 polarization, leads to a significant containment of virus replication. Here we show that M1-MDM restimulation with these cytokines 7 days after infection (M12 MDM) promoted an increased restriction of HIV-1 replication characterized by very low levels of virus production near to undetectable levels. In comparison to control and M1-MDM that were not restimulated, M12 MDM showed a stronger reduction of both total and integrated HIV DNA as well as of viral mRNA expression. M12 MDM were characterized by an upregulated expression of restriction factors acting at the level of reverse transcription (RT), including apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and APOBEC3G, but not SAM domain and HD domain-containing protein 1 (SAMHD1). M12 MDM also showed an increased expression of Class II Transactivator (CIITA) and Tripartite Motif22 (TRIM22), two negative regulators of proviral transcription, whereas expression and phosphorylation of transcriptional inducers of HIV-1, such as nuclear factor kB (NF-kB) and signal transducer and activator of transcription 1 (STAT1), were not impaired in these cells. The almost quiescent state of the infection in M12 MDM was promptly reversed by coculture with mitogen-stimulated leukocytes or cell incubation with their filtered culture supernatant. M12 MDM harbored replication-competent HIV-1 as virus spreading following cell stimulation was fully prevented by the RT inhibitor lamivudine/3TC. Selective reactivation of proviral expression in M12 MDM, but not in control or in M1-MDM that were not restimulated, was confirmed in cells infected with single round Vesicular Stomatitis Virus-G-pseudotyped HIV-1. Thus, M12 MDM represent an in vitro model of reversible, almost quiescent HIV-1 infection of primary human macrophages that could be further exploited for “Cure” related investigations.

SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells.IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.

SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response.

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigen-presenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4+ T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1’s role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

The impact of p53 on the early stage replication of retrovirus

BackgroundThe function of p53 in cancer biology has been studied extensively, but its role in anti-retrovirus infection has been elusive for many years. The restriction of retrovirus early stage replication by p53 was investigated in this study.MethodVSV-G pseudotyped retrovirus with GFP reporter gene was used to infect both HCT116 p53+/+ cells and its isogenic p53 knockout HCT116 p53−/− cells. The infection was detected by flow cytometry. Reverse transcription products were quantified by real time PCR. Mutation analysis was performed after 1-LTR cycle and 2-LTR cycle DNA were amplified and PCR products were sequenced. Transcription and translation of cyclin-dependent kinase inhibitor 1 (p21Cip1) and SAM domain and HD domain-containing protein 1 (SAMHD1) were analyzed by TaqMan PCR and Western blot experiments. siRNA experiment was applied to study the role of p53 downstream gene p21Cip1 in the restriction of retrovirus infection.ResultsIt was found that the block of retrovirus infection in non-cycling cells was significantly attenuated in HCT116 p53−/− cells when compared to HCT116 p53+/+ cells. It was found that both late reverse transcription products and viral 2-LTR cycle DNA were significantly increased in infected non-cycling HCT116 p53−/− cells. Furthermore, the mutation frequency detected in 1-LTR DNA from HCT116 p53+/+ cells were significantly decreased in comparison to HCT116 p53−/− cells. A higher number of insertion and deletion mutations were detected in the joint region of 2-LTR cycle DNA in infected p53+/+ cells. Cell cycle analysis showed retrovirus infection promoted host cell replication. Higher levels of mRNA and protein of p21Cip1 were found in HCT116 p53+/+ cells in comparison to the HCT116 p53−/− cells. Furthermore, knockdown of p21Cip1 in non-cycling HCT116 p53+/+ cells significantly increased the infection.ConclusionsThe results of this study showed that p53 is an important restriction factor that interferes with retrovirus infection in its early stage of replication. Our results suggested that p53 mediates the inhibition of retrovirus infection in non-cycling cells through it downstream gene p21Cip1, and p53 also functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA.