RESUMO
Distinguishing the self from the non-self by the immune system is essential to avoid inflammatory and autoimmune diseases. Maharana et al. (2022) reveal a mechanism for hiding self-immunostimulatory RNA involving a three-variable equation: SAMHD1 and its exonuclease activity, single-stranded RNA, and RNA-protein condensate.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , RNA , Proteína 1 com Domínio SAM e Domínio HD , RNA/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , ExonucleasesRESUMO
Not only does Marseillevirus bear the name of the city where it was identified, it also encompasses its values and what makes Marseille a wonderful city. Marseillevirus is unique and intriguing. As such, Bryson et al. in this issue of Molecular Cell reveal how virion-associated Marseillevirus DNA is packed with nucleosomes.
Assuntos
DNA , Nucleossomos , Nucleossomos/genética , DNA/genética , Vírion/genéticaRESUMO
The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Infecções por HIV/patologia , HIV-1/metabolismo , Imunidade Inata , Complexos Multiproteicos/metabolismo , Recombinases/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , Células HeLa , Humanos , Interferon gama/metabolismoRESUMO
Transcription elongation is increasingly recognized as an important mechanism of gene regulation. Here, we show that microprocessor controls gene expression in an RNAi-independent manner. Microprocessor orchestrates the recruitment of termination factors Setx and Xrn2, and the 3'-5' exoribonuclease, Rrp6, to initiate RNAPII pausing and premature termination at the HIV-1 promoter through cleavage of the stem-loop RNA, TAR. Rrp6 further processes the cleavage product, which generates a small RNA that is required to mediate potent transcriptional repression and chromatin remodeling at the HIV-1 promoter. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), we identified cellular gene targets whose transcription is modulated by microprocessor. Our study reveals RNAPII pausing and premature termination mediated by the co-operative activity of ribonucleases, Drosha/Dgcr8, Xrn2, and Rrp6, as a regulatory mechanism of RNAPII-dependent transcription elongation.
Assuntos
Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Viral da Expressão Gênica , HIV-1/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , DNA Helicases , Repetição Terminal Longa de HIV , Humanos , Dados de Sequência Molecular , Enzimas Multifuncionais , Regiões Promotoras Genéticas , Interferência de RNA , RNA Viral/química , RNA Viral/genética , Fatores de Transcrição/metabolismoRESUMO
Chikungunya virus (CHIKV) is a re-emerging alphavirus that is transmitted to humans by mosquito bites and causes musculoskeletal and joint pain1,2. Despite intensive investigations, the human cellular factors that are critical for CHIKV infection remain unknown, hampering the understanding of viral pathogenesis and the development of anti-CHIKV therapies. Here we identified the four-and-a-half LIM domain protein 1 (FHL1)3 as a host factor that is required for CHIKV permissiveness and pathogenesis in humans and mice. Ablation of FHL1 expression results in the inhibition of infection by several CHIKV strains and o'nyong-nyong virus, but not by other alphaviruses and flaviviruses. Conversely, expression of FHL1 promotes CHIKV infection in cells that do not normally express it. FHL1 interacts directly with the hypervariable domain of the nsP3 protein of CHIKV and is essential for the replication of viral RNA. FHL1 is highly expressed in CHIKV-target cells and is particularly abundant in muscles3,4. Dermal fibroblasts and muscle cells derived from patients with Emery-Dreifuss muscular dystrophy that lack functional FHL15 are resistant to CHIKV infection. Furthermore, CHIKV infection is undetectable in Fhl1-knockout mice. Overall, this study shows that FHL1 is a key factor expressed by the host that enables CHIKV infection and identifies the interaction between nsP3 and FHL1 as a promising target for the development of anti-CHIKV therapies.
Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/patogenicidade , Fatores Celulares Derivados do Hospedeiro/metabolismo , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Animais , Células Cultivadas , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Vírus Chikungunya/crescimento & desenvolvimento , Feminino , Fibroblastos/virologia , Células HEK293 , Fatores Celulares Derivados do Hospedeiro/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/deficiência , Proteínas com Domínio LIM/genética , Masculino , Camundongos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Mioblastos/virologia , Vírus O'nyong-nyong/crescimento & desenvolvimento , Vírus O'nyong-nyong/patogenicidade , Ligação Proteica , RNA Viral/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.
Assuntos
DNA/imunologia , Herpesvirus Humano 8/imunologia , Imunidade Inata , RNA Longo não Codificante/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/imunologia , Autoantígeno Ku/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Complexos Multiproteicos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/imunologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/imunologia , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/genética , Fator de Processamento Associado a PTB/imunologia , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição , TransfecçãoRESUMO
SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.
Assuntos
HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismo , Replicação Viral , Animais , Linhagem Celular , Humanos , Espaço Intracelular/metabolismo , Macaca mulatta , Macrófagos/imunologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteína 1 com Domínio SAM e Domínio HDRESUMO
SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
Assuntos
Replicação do DNA , Interferon Tipo I/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Citosol/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interferon Tipo I/imunologia , Proteína Homóloga a MRE11/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , RecQ Helicases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/deficiênciaRESUMO
This corrects the article DOI: 10.1038/nature21710.
RESUMO
The persistence of the HIV reservoir in infected individuals is a major obstacle to the development of a cure for HIV. Here, using an in vitro model of HIV-infected quiescent CD4 T cells, we reveal a gene expression signature of 103 upregulated genes that are specific for latently infected cells, including genes for 16 transmembrane proteins. In vitro screening for surface expression in HIV-infected quiescent CD4 T cells shows that the low-affinity receptor for the immunoglobulin G Fc fragment, CD32a, is the most highly induced, with no detectable expression in bystander cells. Notably, productive HIV-1 infection of T-cell-receptor-stimulated CD4 T cells is not associated with CD32a expression, suggesting that a quiescence-dependent mechanism is required for its induction. Using blood samples from HIV-1-positive participants receiving suppressive antiretroviral therapy, we identify a subpopulation of 0.012% of CD4 T cells that express CD32a and host up to three copies of HIV DNA per cell. This CD32a+ reservoir was highly enriched in inducible replication-competent proviruses and can be predominant in some participants. Our discovery that CD32a+ lymphocytes represent the elusive HIV-1 reservoir may lead to insights that will facilitate the specific targeting and elimination of this reservoir.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Provírus/crescimento & desenvolvimento , Receptores de IgG/metabolismo , Replicação Viral , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Divisão Celular , Separação Celular , Células Cultivadas , DNA Viral/análise , Perfilação da Expressão Gênica , Células HEK293 , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Provírus/genética , Provírus/isolamento & purificação , Regulação para Cima/genética , Latência Viral/efeitos dos fármacos , Latência Viral/genética , Latência Viral/imunologiaRESUMO
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5' end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Viral/genética , Infecções por HIV/genética , HIV-1/genética , Histonas/genética , Nucleossomos/genética , Integração Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Infecções por HIV/virologia , Humanos , Sequências Repetidas Terminais , Transcrição GênicaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1004343.].
RESUMO
SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Infecções por Herpesviridae/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Antivirais/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Citomegalovirus/genética , Citoplasma/metabolismo , Citoplasma/virologia , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Replicação Viral/efeitos dos fármacosRESUMO
Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.
Assuntos
Proteínas Oncogênicas/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos SCID , Modelos Genéticos , Proteínas Oncogênicas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Ligação Proteica , Interferência de RNA , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transplante HeterólogoRESUMO
Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.
Assuntos
Vírus Chikungunya/fisiologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Pele/patologia , Replicação Viral/fisiologia , Zika virus/fisiologia , Linhagem Celular , Febre de Chikungunya/virologia , Humanos , Anotação de Sequência Molecular , Mapas de Interação de Proteínas , Proteólise , Regulação para Cima , Proteínas Virais Reguladoras e Acessórias/metabolismo , Infecção por Zika virus/virologiaRESUMO
HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Assuntos
Núcleo Celular/metabolismo , HIV-1/genética , RNA Viral/biossíntese , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Linhagem Celular , Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , HIV-1/metabolismo , Células HeLa , Histona-Lisina N-Metiltransferase , Humanos , Complexos Multiproteicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Estresse Fisiológico , Fatores de Transcrição , Fatores de Elongação da Transcrição/metabolismo , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.
Assuntos
Células Dendríticas/metabolismo , HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células Mieloides/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Linhagem Celular , DNA Viral/metabolismo , Células Dendríticas/virologia , Inativação Gênica , Infecções por HIV/metabolismo , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Células Mieloides/virologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Células U937 , Replicação ViralRESUMO
Hepatitis B virus infection (HBV) is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA) that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1). Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection.
Assuntos
Antivirais/metabolismo , Carcinoma Hepatocelular/imunologia , Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/genética , Células Cultivadas , DNA Viral/genética , Hepatite B/metabolismo , Hepatite B/virologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Imunoprecipitação , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Proteínas Associadas aos Microtúbulos/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação ViralRESUMO
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.