RESUMEN
Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.
Asunto(s)
Interferón Tipo I , ARN Bicatenario , Antivirales , Enfermedades Autoinmunes del Sistema Nervioso , Exonucleasas/genética , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , Malformaciones del Sistema Nervioso , ARN Bicatenario/genética , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
Pathogens have fueled the diversification of intracellular defense strategies that collectively define cell-autonomous innate immunity. In bacteria, innate immunity is manifested by a broad arsenal of defense systems that provide protection against bacterial viruses, called phages. The complexity of the bacterial immune repertoire has only been realized recently and is now suggesting that innate immunity has commonalities across the tree of life: many components of eukaryotic innate immunity are found in bacteria where they protect against phages, including the cGAS-STING pathway, gasdermins, and viperins. Here, I summarize recent findings on the conservation of innate immune pathways between prokaryotes and eukaryotes and hypothesize that bacterial defense mechanisms can catalyze the discovery of novel molecular players of eukaryotic innate immunity.
Asunto(s)
Bacterias , Inmunidad Innata , Humanos , Nucleotidiltransferasas/metabolismoRESUMEN
Mutations in sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) are found in a neurodevelopmental disorder, Aicardi-Goutières syndrome, and cancers, and SAMHD1, which is a deoxynucleoside triphosphate (dNTP) triphosphorylase, was identified as a myeloid-specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of an SAMHD1 ortholog of Caenorhabditis elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and triphosphate moieties of dNTPs but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except that its lack of the canonical sterile alpha motif domain. This striking conservation in structure, function, and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.
RESUMEN
HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.
Asunto(s)
VIH-1 , Proteínas de Unión al GTP Monoméricas , Nucleótidos , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Reguladoras y Accesorias Virales , Animales , Humanos , VIH-1/metabolismo , Lentivirus/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1 cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1 cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis.
Asunto(s)
Infecciones por VIH , Interferón Tipo I , Proteína 1 que Contiene Dominios SAM y HD , Humanos , Células HEK293 , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Infecciones por VIH/metabolismo , Transducción de SeñalRESUMEN
Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKß) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/ß/γ via interaction with IKKα and IKKß, thus inhibiting subsequent phosphorylation of IκBα in monocytic THP-1 cells and differentiated nondividing THP-1 cells. We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKß, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/ß/γ in Sendai virus-infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKß in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKß directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKß and that the kinase domain of IKKα and the ubiquitin-like domain of IKKß are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKß. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.
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Quinasa I-kappa B , Proteína 1 que Contiene Dominios SAM y HD , Virosis , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Virosis/inmunología , Virosis/metabolismo , Línea CelularRESUMEN
Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.
Asunto(s)
Reparación del ADN , Desoxirribonucleótidos/metabolismo , Cambio de Clase de Inmunoglobulina , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Línea Celular , Desoxirribonucleótidos/genética , Humanos , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Proteína 1 que Contiene Dominios SAM y HD , Telomerasa , Humanos , Nucleótidos de Desoxiguanina , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
Aicardi-Goutières syndrome (AGS) is an autosomal recessive inflammatory syndrome that manifests as an early-onset encephalopathy with both neurologic and extraneurologic clinical findings. AGS has been associated with pathogenic variants in nine genes: TREX1, RNASEH2B, RNASEH2C, RNASEH2A, SAMHD1, ADAR, IFIH1, LSM11, and RNU7-1. Diagnosis is established by clinical findings (encephalopathy and acquired microcephaly, intellectual and physical impairments, dystonia, hepatosplenomegaly, sterile pyrexia, and/or chilblains), characteristic abnormalities on cranial CT (calcification of the basal ganglia and white matter) and MRI (leukodystrophic changes), or the identification of pathogenic/likely pathogenic variants in the known genes. One of the genes associated with AGS, SAMHD1, has also been associated with a spectrum of cerebrovascular diseases, including moyamoya disease (MMD). In this report, we describe a 31-year-old male referred to genetics for MMD since childhood who lacked the hallmark features of AGS patients but was found to have compound heterozygous SAMHD1 variants. He later developed mitral valve insufficiency due to recurrent chordal rupture and ultimately underwent a heart transplant at 37 years of age. Thus, these data suggest that SAMHD1 pathogenic variants can cause MMD without typical AGS symptoms and support that SAMHD1 should be assessed in MMD patients even in the absence of AGS features.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Encefalopatías , Enfermedad de Moyamoya , Malformaciones del Sistema Nervioso , Masculino , Humanos , Niño , Adulto , Proteína 1 que Contiene Dominios SAM y HD/genética , Enfermedad de Moyamoya/complicaciones , Válvula Mitral/patología , Mutación , Malformaciones del Sistema Nervioso/diagnóstico por imagen , Malformaciones del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/diagnóstico , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/patología , Encefalopatías/complicacionesRESUMEN
BACKGROUND: Influenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV. METHODS: To investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication. RESULTS: In this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1's ability to restrict H5N1 is related to phosphorylation at 592-tyrosine. CONCLUSIONS: In conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Animales , Humanos , Virus de la Influenza A/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Replicación Viral , Proteínas Virales/metabolismo , Antivirales/farmacología , Antivirales/metabolismo , Factor 3 Regulador del Interferón/metabolismoRESUMEN
The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi-Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR-Cas9 gene KO or lentiviral viral protein X-mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2-infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.
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COVID-19 , Proteína 1 que Contiene Dominios SAM y HD , SARS-CoV-2 , Antivirales/farmacología , Enfermedades Autoinmunes del Sistema Nervioso , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Humanos , Inmunidad Innata , Interferones , Malformaciones del Sistema Nervioso , ARN Viral , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , Replicación Viral/inmunologíaRESUMEN
BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.
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VIH-1 , Proteínas de Unión al GTP Monoméricas , Humanos , VIH-1/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Fosforilación , Células U937 , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is a sphingolipid modulator of a myriad of cellular processes, and therapeutic targeting of S1P signaling is utilized clinically to treat multiple sclerosis. We have previously shown that functional antagonism of S1P receptors reduces cell-free, cell-to-cell, and latent HIV-1 infection in primary CD4 T cells. In this work, we examined whether targeting sphingosine kinase 1 or 2 (SPHK1/2) to inhibit S1P production would prevent infection using multiple HIV-1 primary isolates and infectious molecular clones. SPHK inhibition reduced HIV transmission between primary CD4 T cells in both cell-to-cell transmission and pretreatment coculture models. Mechanistically, pharmacological inhibition of SPHK reduced susceptibility to infection primarily by downregulating phosphorylated SAMHD1 (pSAMHD1), enhancing the activity of this innate HIV-1 restriction factor. Furthermore, genetic disruption of either SPHK1 or SPHK2 by CRISPR/Cas9 reduced phosphorylation of SAMHD1, demonstrating the role of these kinases in modulation of SAMHD1 activity. The effect of SPHK inhibition on limiting HIV-1 infection in CD4 T cells was observed irrespective of the biological sex or age of the donor, with neither variable significantly influencing the effectiveness of SPHK inhibition. Our results demonstrate that targeting SPHK inhibits transmission of HIV-1 via modulation of SAMHD1 phosphorylation to decrease permissiveness to infection in CD4 T cells and suggests that therapeutic targeting of this pathway early in infection enables development of strategies to prevent establishment of infection and hinder cell-to-cell transmission of HIV-1. IMPORTANCE HIV-1 infection, once established, requires lifelong treatment due to the ability of the virus to maintain latent infection in its host and become reactivated during an interruption in antiretroviral treatment (ART). Although preventing transmission and acquisition of HIV is an important goal, no ART thus far have exploited harnessing a component of the host immune system to combat transmission of the virus. We have previously shown that inhibition of sphingosine-1-phosphate (S1P) receptors, a component of S1P signaling, reduces HIV-1 infection in human CD4 T cells. We therefore investigated inhibition of sphingosine kinases, another element of this signaling system, in this work. We found that inhibition of sphingosine kinases 1 and 2 (SPHK1/2) could reduce HIV-1 transmission, both among CD4 T cells and between macrophages and CD4 T cells. Our research therefore suggests that therapeutic targeting of SPHK or S1P receptors may aid in the development of strategies to prevent establishment and transmission of HIV-1 infection among immune cells.
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Linfocitos T CD4-Positivos , Infecciones por VIH , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteína 1 que Contiene Dominios SAM y HD , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Humanos , Lisofosfolípidos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Latencia del VirusRESUMEN
The cellular protein SAMHD1 is important for DNA repair, suppressing LINE elements, controlling deoxynucleoside triphosphate (dNTP) concentrations, maintaining HIV-1 latency, and preventing excessive type I interferon responses. SAMHD1 is also a potent inhibitor of HIV-1 and other significant viral pathogens. Infection restriction is due in part to the deoxynucleoside triphosphatase (dNTPase) activity of SAMHD1 but is also mediated through a dNTPase-independent mechanism that has been described but not explored. The phosphorylation of SAMHD1 at threonine 592 (T592) controls many of its functions. Retroviral restriction, irrespective of dNTPase activity, is linked to unphosphorylated T592. Sulforaphane (SFN), an isothiocyanate, protects macrophages from HIV infection by mobilizing the transcription factor and antioxidant response regulator Nrf2. Here, we show that SFN and other clinically relevant Nrf2 mobilizers reduce SAMHD1 T592 phosphorylation to protect macrophages from HIV-1. We further show that SFN, through Nrf2, triggers the upregulation of the cell cycle control protein p21 in human monocyte-derived macrophages to contribute to SAMHD1 activation. We additionally present data that support another, potentially redox-dependent mechanism employed by SFN to contribute to SAMHD1 activation through reduced phosphorylation. This work establishes the use of exogenous Nrf2 mobilizers as a novel way to study virus restriction by SAMHD1 and highlights the Nrf2 pathway as a potential target for the therapeutic control of SAMHD1 cellular and antiviral functions. IMPORTANCE Here, we show, for the first time, that the treatment of macrophages with Nrf2 mobilizers, known activators of antioxidant responses, increases the fraction of SAMHD1 without a regulatory phosphate at position 592. We demonstrate that this decreases infection of macrophages by HIV-1. Phosphorylated SAMHD1 is important for DNA repair, the suppression of LINE elements, the maintenance of HIV-1 in a latent state, and the prevention of excessive type I interferon responses, while unphosphorylated SAMHD1 blocks HIV infection. SAMHD1 impacts many viruses and is involved in various cancers, so knowledge of how it works and how it is regulated has broad implications for the development of therapeutics. Redox-modulating therapeutics are already in clinical use or under investigation for the treatment of many conditions. Thus, understanding the impact of redox modifiers on controlling SAMHD1 phosphorylation is important for many areas of research in microbiology and beyond.
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Infecciones por VIH , Interferón Tipo I , Isotiocianatos , Proteína 1 que Contiene Dominios SAM y HD , Humanos , Antioxidantes/metabolismo , VIH-1 , Interferón Tipo I/metabolismo , Isotiocianatos/farmacología , Macrófagos , Factor 2 Relacionado con NF-E2/metabolismo , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD/metabolismoRESUMEN
Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.
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Nefritis Lúpica , FN-kappa B , Humanos , Células Cultivadas , Interferón gamma/metabolismo , Nefritis Lúpica/genética , Células Mesangiales/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/farmacología , Receptor de Interferón gammaRESUMEN
Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1-/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Inestabilidad Genómica/fisiología , Ribonucleótidos/genética , Ribonucleótidos/metabolismo , Animales , Daño del ADN , Femenino , Dosificación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
Over-activation of Toll-like receptor 4 (TLR4) is the key mechanism in Gram-negative bacterial infection-induced sepsis. SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) inhibits multiple viruses, but whether it plays a role during bacterial invasion remains unelucidated. Monocyte-macrophage specific Samhd1 knockout (Samhd1-/-) mice and Samhd1-/- macrophage cell line RAW264.7 were constructed and used as research models to evaluate the role of SAMHD1 in TLR4-activated inflammation. In vivo, LPS-challenged Samhd1-/- mice showed higher serum inflammatory factors, accompanied with more severe inflammation infiltration and lower survival rate. In vitro, Samhd1-/- peritoneal macrophages had more activated TLR4 pathway upon LPS-stimulation, accompanied with mitochondrial depolarization and dysfunction and a higher tendency to be M1-polarized. These results could be rescued by overexpressing full-length wild-type SAMHD1 or its phospho-mimetic T634D mutant into Samhd1-/- RAW264.7 cells, whereas the mutants, dNTP hydrolase-function-deprived H238A and phospho-ablative T634A, did not exert the same effect. Lastly, co-IP and immunofluorescence assays confirmed that SAMHD1 interacted with an outer mitochondrial membrane-localized protein, voltage-dependent anion channel-1 (VDAC1). SAMHD1 inhibits TLR4-induced acute inflammation and M1 polarization of macrophages by interacting with VDAC1 and maintaining mitochondria function, which outlines a novel regulatory mechanism of TLR signaling upon LPS stimulation.
Asunto(s)
Lipopolisacáridos , Receptor Toll-Like 4 , Animales , Ratones , Inflamación , Lipopolisacáridos/toxicidad , Macrófagos , Mitocondrias , Proteína 1 que Contiene Dominios SAM y HD/genética , Receptor Toll-Like 4/genéticaRESUMEN
Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.
Asunto(s)
Neoplasias del Colon , Leucemia , Mutación Missense , Proteínas de Neoplasias , Proteína 1 que Contiene Dominios SAM y HD , Sustitución de Aminoácidos , Línea Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Ciclina A2/química , Ciclina A2/genética , Ciclina A2/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Leucemia/enzimología , Leucemia/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/química , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: TASOR, a component of the HUSH repressor epigenetic complex, and SAMHD1, a cellular triphosphohydrolase (dNTPase), are both anti-HIV proteins antagonized by HIV-2/SIVsmm Viral protein X. As a result, the same viral protein is able to relieve two different blocks along the viral life cell cycle, one at the level of reverse transcription, by degrading SAMHD1, the other one at the level of proviral expression, by degrading TASOR. Phosphorylation of SAMHD1 at T592 has been shown to downregulate its antiviral activity. The discovery that T819 in TASOR was lying within a SAMHD1 T592-like motif led us to ask whether TASOR is phosphorylated on this residue and whether this post-translational modification could regulate its repressive activity. RESULTS: Using a specific anti-phospho-antibody, we found that TASOR is phosphorylated at T819, especially in cells arrested in early mitosis by nocodazole. We provide evidence that the phosphorylation is conducted by a Cyclin/CDK1 complex, like that of SAMHD1 at T592. While we could not detect TASOR in quiescent CD4 + T cells, TASOR and its phosphorylated form are present in activated primary CD4 + T lymphocytes. In addition, TASOR phosphorylation appears to be independent from TASOR repressive activity. Indeed, on the one hand, nocodazole barely reactivates HIV-1 in the J-Lat A1 HIV-1 latency model despite TASOR T819 phosphorylation. On the other hand, etoposide, a second cell cycle arresting drug, reactivates latent HIV-1, without concomitant TASOR phosphorylation. Furthermore, overexpression of wt TASOR or T819A or T819E similarly represses gene expression driven by an HIV-1-derived LTR promoter. Finally, while TASOR is degraded by HIV-2 Vpx, TASOR phosphorylation is prevented by HIV-1 Vpr, likely as a consequence of HIV-1 Vpr-mediated-G2 arrest. CONCLUSIONS: Altogether, we show that TASOR phosphorylation occurs in vivo on T819. This event does not appear to correlate with TASOR-mediated HIV-1 silencing. We speculate that TASOR phosphorylation is related to a role of TASOR during cell cycle progression.
Asunto(s)
Infecciones por VIH , VIH-1 , Proteínas de Unión al GTP Monoméricas , Humanos , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , VIH-1/fisiología , Fosforilación , Treonina , Nocodazol/metabolismo , Latencia del Virus , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs.