RESUMO
SAMHD1 is a potent HIV-1 restriction factor that blocks reverse transcription in monocytes, dendritic cells and resting CD4+ T cells by decreasing intracellular dNTP pools. However, SAMHD1 may diminish innate immune sensing and Ag presentation, resulting in a weaker adaptive immune response. To date, the role of SAMHD1 on antiretroviral immunity remains unclear, as mouse SAMHD1 had no impact on murine retrovirus replication in prior in vivo studies. Here, we show that SAMHD1 significantly inhibits acute Friend retrovirus infection in mice. Pretreatment with LPS, a significant driver of inflammation during HIV-1 infection, further unmasked a role for SAMHD1 in influencing immune responses. LPS treatment in vivo doubled the intracellular dNTP levels in immune compartments of SAMHD1 knockout but not wild-type mice. SAMHD1 knockout mice exhibited higher plasma infectious viremia and proviral DNA loads than wild-type mice at 7 d postinfection (dpi), and proviral loads inversely correlated with a stronger CD8+ T cell response. SAMHD1 deficiency was also associated with weaker NK, CD4+ T and CD8+ T cell responses by 14 dpi and weaker neutralizing Ab responses by 28 dpi. Intriguingly, SAMHD1 influenced these cell-mediated immune (14 dpi) and neutralizing Ab (28 dpi) responses in male but not female mice. Our findings formally demonstrate SAMHD1 as an antiretroviral factor in vivo that could promote adaptive immune responses in a sex-dependent manner. The requirement for LPS to unravel the SAMHD1 immunological phenotype suggests that comorbidities associated with a "leaky" gut barrier may influence the antiviral function of SAMHD1 in vivo.
Assuntos
Imunidade Adaptativa/imunologia , Vírus da Leucemia Murina de Friend/crescimento & desenvolvimento , Lipopolissacarídeos/farmacologia , Infecções por Retroviridae/prevenção & controle , Proteína 1 com Domínio SAM e Domínio HD/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , DNA Viral/sangue , Feminino , Vírus da Leucemia Murina de Friend/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Retroviridae/virologia , Transcrição Reversa/genética , Proteína 1 com Domínio SAM e Domínio HD/imunologia , Carga ViralRESUMO
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.
Assuntos
COVID-19 , Proteína 1 com Domínio SAM e Domínio HD , SARS-CoV-2 , Antivirais/farmacologia , Doenças Autoimunes do Sistema Nervoso , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Humanos , Imunidade Inata , Interferons , Malformações do Sistema Nervoso , RNA Viral , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral/imunologiaRESUMO
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.
Assuntos
Neoplasias do Colo , Leucemia , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Proteína 1 com Domínio SAM e Domínio HD , Substituição de Aminoácidos , Linhagem Celular , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Ciclina A2/química , Ciclina A2/genética , Ciclina A2/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Leucemia/enzimologia , Leucemia/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Relação Estrutura-AtividadeRESUMO
Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.
Assuntos
Amidoidrolases/genética , Transformação Celular Neoplásica/genética , Replicação do DNA/genética , Transcrição Gênica , Animais , Antineoplásicos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Uracila/análogos & derivados , Uracila/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1's restriction of dNTP supply enhances AID's mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.
Assuntos
Linfócitos B/imunologia , Fase G1/imunologia , Ativação Linfocitária , Mutação , Proteína 1 com Domínio SAM e Domínio HD , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Linfócitos B/citologia , Citidina Desaminase/imunologia , DNA Polimerase Dirigida por DNA , Fase G1/genética , Masculino , Camundongos , Camundongos Transgênicos , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/imunologiaRESUMO
BACKGROUND: Terminally 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. RESULTS: The 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. CONCLUSION: Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.
Assuntos
Desoxirribonucleotídeos/farmacologia , HIV-1/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Transcrição Reversa/efeitos dos fármacos , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Células Cultivadas , HIV-1/genética , Humanos , Cinética , Replicação Viral/efeitos dos fármacosRESUMO
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.
Assuntos
Infecções por HIV , HIV-1 , Mutação de Sentido Incorreto , Multimerização Proteica , Proteína 1 com Domínio SAM e Domínio HD , Motivos de Aminoácidos , Substituição de Aminoácidos , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Relação Estrutura-Atividade , Células THP-1 , Células U937RESUMO
The triphosphohydrolase SAMHD1 (sterile α motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication.
Assuntos
Infecções por HIV/enzimologia , HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Multimerização Proteica , RNA Viral/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Infecções por HIV/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Fosforilação , RNA Viral/genética , Proteína 1 com Domínio SAM e Domínio HD , Células U937RESUMO
The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg(2+) or Mn(2+) ions, which are critical for its catalytic function. There is great interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We focused here on a series of ß-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, in order to build a three-dimensional pharmacophore model. In addition, we developed a molecular beacon (MB)-based PA-Nter assay that enabled us to compare the inhibition of Mn(2+) versus Mg(2+), the latter probably being the biologically relevant cofactor. This real-time MB assay allowed us to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC50 values comparable to that of the prototype L-742,001 (i.e., below 2 µM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg(2+) or Mn(2+). Three compounds ( 10: , with a pyrrole scaffold, and 40: and 41: , with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values 64-95 µM) and were proven to affect viral RNA synthesis. Our approach of integrating complementary enzymatic, cellular, and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.
Assuntos
Antivirais/farmacologia , Quelantes/farmacologia , Descoberta de Drogas/métodos , Endonucleases/antagonistas & inibidores , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/enzimologia , Animais , Antivirais/química , Quelantes/química , Cães , Endonucleases/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Conformação MolecularRESUMO
All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of "alarmone" (p)ppGpp to arrest proliferation and reprogram transcriptome. While mammalian genomes encode MESH1-the homolog of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumor sphere formation, and retarded xenograft growth. These antitumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed expression of TAZ, a HIPPO coactivator, and proliferative gene. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, reduced sphere formation, tumor growth inhibition, dNTP depletion, and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.
Assuntos
Guanosina Pentafosfato , Fatores de Transcrição , Acetilação , Animais , Proliferação de Células/genética , Humanos , Mamíferos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The restriction factor SAMHD1 regulates intracellular nucleotide level by degrading dNTPs and blocks the replication of retroviruses and DNA viruses in non-cycling cells, like macrophages or dendritic cells. In patients, inactivating mutations in samhd1 are associated with the autoimmune disease Aicardi-Goutières Syndrome (AGS). The accumulation of intracellular nucleic acids derived from endogenous retroelements thriving in the absence of SAMHD1 has been discussed as potential trigger of the autoimmune reaction. In vitro, SAMHD1 has been found to restrict endogenous retroelements, like LINE-1 elements (L1). The mechanism, however, by which SAMHD1 blocks endogenous retroelements, is still unclear. RESULTS: Here, we show that SAMHD1 inhibits the replication of L1 and other endogenous retroelements in cycling cells. By applying GFP- and neomycin-based reporter assays we found that the anti-L1 activity of SAMHD1 is regulated by phosphorylation at threonine 592 (T592). Similar to the block of HIV, the cofactor binding site and the enzymatic active HD domain of SAMHD1 proofed to be essential for restriction of L1 elements. However, phosphorylation at T592 did not correlate with the dNTP hydrolase activity of SAMHD1 in cycling 293T cells suggesting an alternative mechanism of regulation. Interestingly, we found that SAMHD1 binds to ORF2 protein of L1 and that this interaction is regulated by T592 phosphorylation. Together with the finding that the block is also active in cycling cells, our results suggest that the SAMHD1-mediated inhibition of L1 is similar but not identical to HIV restriction. CONCLUSION: Our findings show conclusively that SAMHD1 restricts the replication of endogenous retroelements in vitro. The results suggest that SAMHD1 is important for maintaining genome integrity and support the idea of an enhanced replication of endogenous retroelements in the absence of SAMHD1 in vivo, potentially triggering autoimmune diseases like AGS. Our analysis also contributes to the better understanding of the activities of SAMHD1 in antiviral defense and nucleotide metabolism. The finding that the phosphorylation of SAMHD1 at T592 regulates its activity against retroelements but not necessarily intracellular dNTP level suggests that the dNTP hydrolase activity might not be the only function of SAMHD1 important for its antiviral activity and for controlling autoimmunity.
RESUMO
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
SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4+ T cells enter the G1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.
Assuntos
Holoenzimas/metabolismo , Mitose , Proteína Fosfatase 2/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Células HEK293 , HIV-1/fisiologia , Células HeLa , Holoenzimas/genética , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/genética , Interferência de RNA , Proteína 1 com Domínio SAM e Domínio HD/genética , Células THP-1RESUMO
The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIVmac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Triazinas/farmacologia , Animais , Linhagem Celular , Cães , HIV-1/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Inibidores da Transcriptase Reversa/química , Triazinas/químicaRESUMO
DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.
Assuntos
Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Proteína 1 com Domínio SAM e Domínio HD/genética , Quebras de DNA de Cadeia Dupla , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , TransfecçãoRESUMO
Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Papillomavirus Humano 31/fisiologia , Queratinócitos/virologia , Infecções por Papillomavirus/enzimologia , Ribonucleosídeo Difosfato Redutase/metabolismo , Replicação Viral , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , Replicação do DNA , Desoxirribonucleosídeos/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 31/genética , Humanos , Queratinócitos/enzimologia , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Domínios Proteicos , Ribonucleosídeo Difosfato Redutase/genéticaRESUMO
Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.
Assuntos
Quinase 2 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , HIV-1/patogenicidade , Vírus da Leucemia Murina/patogenicidade , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Proteína Quinase CDC2 , Linhagem Celular , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células HEK293 , Infecções por HIV/patologia , HIV-1/genética , Humanos , Vírus da Leucemia Murina/genética , Leucemia Experimental/patologia , Camundongos , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Infecções por Retroviridae/patologia , Proteína 1 com Domínio SAM e Domínio HD , Transfecção , Infecções Tumorais por Vírus/patologia , Células U937 , Replicação Viral/genéticaRESUMO
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 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G1/G0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Monócitos/metabolismo , Monócitos/virologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Apoptose/genética , Biomarcadores , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Técnicas de Inativação de Genes , Inativação Gênica , Infecções por HIV/genética , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.