RESUMO
Long noncoding RNAs (lncRNAs) can exert their function by interacting with the DNA via triplex structure formation. Even though this has been validated with a handful of experiments, a genome-wide analysis of lncRNA-DNA binding is needed. In this paper, we develop and interpret deep learning models that predict the genome-wide binding sites deciphered by ChIRP-Seq experiments of 12 different lncRNAs. Among the several deep learning architectures tested, a simple architecture consisting of two convolutional neural network layers performed the best suggesting local sequence patterns as determinants of the interaction. Further interpretation of the kernels in the model revealed that these local sequence patterns form triplex structures with the corresponding lncRNAs. We uncovered several novel triplexes forming domains (TFDs) of these 12 lncRNAs and previously experimentally verified TFDs of lncRNAs HOTAIR and MEG3. We experimentally verified such two novel TFDs of lncRNAs HOTAIR and TUG1 predicted by our method (but previously unreported) using Electrophoretic mobility shift assays. In conclusion, we show that simple deep learning architecture can accurately predict genome-wide binding sites of lncRNAs and interpretation of the models suggest RNA:DNA:DNA triplex formation as a viable mechanism underlying lncRNA-DNA interactions at genome-wide level.
Assuntos
Sítios de Ligação , Aprendizado Profundo , Estudo de Associação Genômica Ampla , RNA Longo não Codificante/genética , Animais , DNA , Regulação da Expressão Gênica , Humanos , Camundongos , Reprodutibilidade dos TestesRESUMO
Mutations in the human SAMHD1 gene are known to correlate with the development of the Aicardi-Goutières syndrome (AGS), which is an inflammatory encephalopathy that exhibits neurological dysfunction characterized by increased production of type I interferon (IFN); this evidence has led to the concept that the SAMHD1 protein negatively regulates the type I IFN response. Additionally, the SAMHD1 protein has been shown to prevent efficient HIV-1 infection of macrophages, dendritic cells, and resting CD4+ T cells. To gain insights on the SAMHD1 molecular determinants that are responsible for the deregulated production of type I IFN, we explored the biochemical, cellular, and antiviral properties of human SAMHD1 mutants known to correlate with the development of AGS. Most of the studied SAMHD1 AGS mutants exhibit defects in the ability to oligomerize, decrease the levels of cellular deoxynucleotide triphosphates in human cells, localize exclusively to the nucleus, and restrict HIV-1 infection. At least half of the tested variants preserved the ability to be degraded by the lentiviral protein Vpx, and all of them interacted with RNA. Our investigations revealed that the SAMHD1 AGS variant p.G209S preserve all tested biochemical, cellular, and antiviral properties, suggesting that this residue is a determinant for the ability of SAMHD1 to negatively regulate the type I IFN response in human patients with AGS. Overall, our work genetically separated the ability of SAMHD1 to negatively regulate the type I IFN response from its ability to restrict HIV-1.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Infecções por HIV/genética , Interferon Tipo I/genética , Malformações do Sistema Nervoso/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Doenças Autoimunes do Sistema Nervoso/complicações , Doenças Autoimunes do Sistema Nervoso/virologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Predisposição Genética para Doença , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Lentivirus/genética , Mutação , Malformações do Sistema Nervoso/complicações , Malformações do Sistema Nervoso/virologiaRESUMO
DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Replicação Viral/imunologiaRESUMO
UNLABELLED: Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. IMPORTANCE: SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , HIV-1/imunologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Animais , Células Cultivadas , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína 1 com Domínio SAM e Domínio HD , Replicação ViralRESUMO
From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of "RAN holes" void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus.
Assuntos
Lamina Tipo B/metabolismo , Poro Nuclear/metabolismo , Síndrome de Werner/metabolismo , Animais , Western Blotting , Galinhas , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Técnicas de Inativação de Genes , Proteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: The IFN-α-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Genetic evidence suggested that capsid is the viral determinant for restriction by MxB. This work explores the ability of MxB to bind to the HIV-1 core, and the role of capsid-binding in restriction. RESULTS: We showed that MxB binds to the HIV-1 core and that this interaction leads to inhibition of the uncoating process of HIV-1. These results identify MxB as an endogenously expressed protein with the ability to inhibit HIV-1 uncoating. In addition, we found that a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to capsid. The use of this small-molecule identified the MxB binding region on the surface of the HIV-1 core. Domain mapping experiments revealed the following requirements for restriction: 1) MxB binding to the HIV-1 capsid, which requires the 20 N-terminal amino acids, and 2) oligomerization of MxB, which is mediated by the C-terminal domain provides the avidity for the interaction of MxB with the HIV-1 core. CONCLUSIONS: Overall our work establishes that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1. Moreover, we demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas do Core Viral/metabolismo , Capsídeo/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Ligação Proteica , Células U937RESUMO
Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread, but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1 and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in nondividing macrophages.
Assuntos
Replicação do DNA , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linhagem Celular , Regulação para Baixo , Herpes Simples/genética , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteína 1 com Domínio SAM e Domínio HD , Replicação ViralRESUMO
BACKGROUND: Expression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration. RESULTS: This work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection. CONCLUSIONS: These results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.
Assuntos
HIV-1/imunologia , HIV-1/fisiologia , Transcrição Reversa , Integração Viral , beta Carioferinas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Linhagem Celular , HumanosRESUMO
BACKGROUND: SAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction. RESULTS: Structural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109-626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection. CONCLUSIONS: These results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.
Assuntos
HIV-1/imunologia , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Multimerização Proteica , Linhagem Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Imunoprecipitação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HDRESUMO
BACKGROUND: Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) is a recently identified host factor that restricts HIV-1 replication in dendritic and myeloid cells. SAMHD1 is a dNTPase that presumably reduces the cellular dNTP levels to levels too low for retroviral reverse transcription to occur. However, HIV-2 and SIV encoded Vpx counteracts the antiviral effects of SAMHD1 by targeting the protein for proteasomal degradation. SAMHD1 is encoded by a multiply spliced mRNA and consists of 16 coding exons. RESULTS: Here, we identified two naturally occurring splice variants lacking exons 8-9 and 14, respectively. Like wildtype SAMHD1, both splice variants localize primarily to the nucleus, interact with Vpx, and retain some sensitivity to Vpx-dependent degradation. However, the splice variants differ from full-length SAMHD1 in their metabolic stability and catalytic activity. While full-length SAMHD1 is metabolically stable in uninfected cells, both splice variants were inherently metabolically unstable and were rapidly degraded even in the absence of Vpx. Vpx strongly increased the rate of degradation of full-length SAMHD1 and further accelerated the degradation of the splice variants. However, the effect of Vpx on the splice variants was more modest due to the inherent instability of these proteins. Analysis of dNTPase activity indicates that neither splice variant is catalytically active. CONCLUSIONS: The identification of SAMHD1 splice variants exposes a potential regulatory mechanism that could enable the cell to control its dNTPase activity on a post-transcriptional level.
Assuntos
Variação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linhagem Celular , Núcleo Celular/química , Éxons , Humanos , Modelos Moleculares , Estabilidade Proteica , Proteína 1 com Domínio SAM e Domínio HDRESUMO
BACKGROUND: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation. RESULTS: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1. CONCLUSIONS: We mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.
Assuntos
Núcleo Celular/metabolismo , HIV-1/patogenicidade , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sinais de Localização Nuclear/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Síndrome da Imunodeficiência Adquirida/virologia , Transporte Ativo do Núcleo Celular , Alelos , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/metabolismo , Citoplasma/virologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Mutagênese Sítio-Dirigida , Mutação , Proteólise , Mapeamento por Restrição , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Células U937 , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Receptor Patched-1/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas Hedgehog/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Osteoblastos/citologia , Receptor Patched-1/genética , Transdução de SinaisRESUMO
SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.
Assuntos
Cisteína/metabolismo , Retroviridae/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Replicação Viral/fisiologia , Cisteína/genética , Células HEK293 , Humanos , Mutação , Oxirredução , Proteína 1 com Domínio SAM e Domínio HD/genética , Células U937RESUMO
The HIV-1 restriction factor SAMHD1 has the ability to negatively modulate retrotransposition of the long interspersed element 1(LINE-1). By exploring the ability of human SAMHD1 polymorphisms to inhibit LINE-1, we found that the single nucleotide polymorphism S33A present in the Korean population lose the ability to inhibit LINE-1 retrotransposition. Because SAMHD1 residue S33 is phosphorylated in human cycling and non-cycling cells, we demonstrated that SAMHD1 requires to be either phosphorylated on position 33 or to contain a bulky residue in order to inhibit LINE-1 retrotransposition. Therefore this unique mutation uncouples functions in this important restriction factor.
RESUMO
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.
Assuntos
HIV/fisiologia , Nucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Tirosina/genética , Regulação Alostérica , Substituição de Aminoácidos , Linhagem Celular , Humanos , Modelos Moleculares , Mutação , Fosforilação , Multimerização Proteica , Proteína 1 com Domínio SAM e Domínio HD/química , Relação Estrutura-AtividadeRESUMO
SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition.
Assuntos
Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , HIV-2/fisiologia , Proteínas Monoméricas de Ligação ao GTP/genética , Polimorfismo de Nucleotídeo Único , Animais , Desoxirribonucleotídeos/metabolismo , Infecções por HIV/metabolismo , Humanos , Elementos Nucleotídeos Longos e Dispersos , Proteína 1 com Domínio SAM e Domínio HDRESUMO
The HIV-1 restriction factor SAM domain- and HD domain-containing protein 1 (SAMHD1) is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4(+) T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Viral/metabolismo , Replicação Viral , Doenças Autoimunes do Sistema Nervoso/genética , Sequência de Bases , Sítios de Ligação/genética , Linfócitos T CD4-Positivos , Linhagem Celular Tumoral , Infecções por HIV/genética , Células HeLa , Humanos , Macrófagos , Mutação , Malformações do Sistema Nervoso/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/genética , Ribonucleases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Análise de Sequência de RNARESUMO
SAMHD1 is a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and inhibits the ability of retroviruses, notably HIV-1, to infect myeloid cells. Although SAMHD1 is expressed in both cycling and noncycling cells, the antiviral activity of SAMHD1 is limited to noncycling cells. We determined that SAMHD1 is phosphorylated on residue T592 in cycling cells but that this phosphorylation is lost when cells are in a noncycling state. Reverse genetic experiments revealed that SAMHD1 phosphorylated on residue T592 is unable to block retroviral infection, but this modification does not affect the ability of SAMHD1 to decrease cellular dNTP levels. SAMHD1 contains a target motif for cyclin-dependent kinase 1 (cdk1) ((592)TPQK(595)), and cdk1 activity is required for SAMHD1 phosphorylation. Collectively, these findings indicate that phosphorylation modulates the ability of SAMHD1 to block retroviral infection without affecting its ability to decrease cellular dNTP levels.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células Mieloides/metabolismo , Células Mieloides/virologia , Nucleotídeos/genética , Nucleotídeos/metabolismo , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Infecções por Retroviridae/virologia , Proteína 1 com Domínio SAM e Domínio HD , Células U937RESUMO
The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to block HIV-1 and SIV(mac) infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine-aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIV(mac). We demonstrated that the HD domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIV(mac). These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV).
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Infecções por Retroviridae/virologia , Retroviridae/fisiologia , Linhagem Celular , Células Dendríticas/virologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , HIV-1/genética , HIV-1/fisiologia , HIV-2/genética , HIV-2/fisiologia , Células HeLa , Humanos , Vírus da Imunodeficiência Bovina/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Vírus da Anemia Infecciosa Equina/fisiologia , Vírus da Leucemia Murina/fisiologia , Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Proteína 1 com Domínio SAM e Domínio HD , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Células U937RESUMO
The gateway for molecular trafficking between the cytoplasm and the nucleus is the Nuclear Pore Complex (NPC). Through mass spectral analysis of the isolated Nuclear Pore Nup107-160 subcomplex, we discovered an in vivo interaction with Werner's Helicase Interacting Protein 1, (WRNIP1 or WHIP). WHIP was originally identified as a binding partner of Werner protein (WRN), which functions to maintain genome stability and is responsible for the progeria disease, Werner syndrome. We established the reciprocal isolation of Nup107 by alpha-WHIP. WHIP was found in purified Nuclear Envelope (NE) fractions treated with DNase/RNase/Heparin. We demonstrated by immunofluorescence microscopy that WHIP is located at the nuclear rim as well as punctate regions in the nuclear matrix. Ultimately, synchronized cells show a dynamic association between WHIP and the Nup107-160 subcomplex through the cell cycle without an interaction with WRN. We thus identify WHIP as a partner/component of the NE/NPC and set forth to investigate a role for the protein positioned at the NPC.