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1.
Mol Cell ; 67(3): 387-399.e5, 2017 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-28712728

RESUMEN

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.


Asunto(s)
ADN/inmunología , Herpesvirus Humano 8/inmunología , Inmunidad Innata , ARN Largo no Codificante/inmunología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/inmunología , Autoantígeno Ku/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/inmunología , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/inmunología , Factor de Empalme Asociado a PTB/metabolismo , Unión Proteica , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factores de Transcripción , Transfección
2.
Nature ; 557(7703): 57-61, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29670289

RESUMEN

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.


Asunto(s)
Replicación del ADN , Interferón Tipo I/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Citosol/metabolismo , ADN de Cadena Simple/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Interferón Tipo I/inmunología , Proteína Homóloga de MRE11/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , RecQ Helicasas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/deficiencia
3.
Int J Mol Sci ; 20(7)2019 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-30959732

RESUMEN

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.


Asunto(s)
Virus Chikungunya/fisiología , Fibroblastos/metabolismo , Fibroblastos/virología , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Piel/patología , Replicación Viral/fisiología , Virus Zika/fisiología , Línea Celular , Fiebre Chikungunya/virología , Humanos , Anotación de Secuencia Molecular , Mapas de Interacción de Proteínas , Proteolisis , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales/metabolismo , Infección por el Virus Zika/virología
4.
Retrovirology ; 12: 103, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26667483

RESUMEN

BACKGROUND: Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies. RESULTS: We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA. CONCLUSION: Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.


Asunto(s)
VIH-1/fisiología , Virus de la Leucemia Murina/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Infecciones por Retroviridae/virología , Transcripción Reversa , Animales , Línea Celular , Células Cultivadas , Humanos , Macrófagos/virología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Fosforilación , ARN Viral/genética , ARN Viral/metabolismo , Proteína 1 que Contiene Dominios SAM y HD , Treonina/fisiología , Replicación Viral
5.
Retrovirology ; 9: 87, 2012 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-23092122

RESUMEN

BACKGROUND: Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. RESULTS: Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. CONCLUSION: Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , VIH-1/inmunología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Proteínas de Unión al GTP Monoméricas/metabolismo , Transcripción Reversa , ADN Viral/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/inmunología , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Reguladoras y Accesorias Virales/inmunología , Proteínas Reguladoras y Accesorias Virales/metabolismo
6.
Retrovirology ; 8: 104, 2011 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-22176773

RESUMEN

BACKGROUND: Integration of human immunodeficiency virus type 1 (HIV-1) into a host cell chromosome is an essential step under the control of the viral integrase (IN). Although this enzyme is necessary and sufficient to catalyze the integration reaction in vitro, cellular cofactors are involved in the process in vivo. The chromatin-associated factor LEDGF/p75 interacts with IN and promotes integration to transcription units of the host genome. HIV-1 IN also binds the karyopherin TNPO3, however the significance of this interaction during viral replication remains to be explored. RESULTS: Here we present a functional analysis of IN mutants impaired for LEDGF/p75 and TNPO3 interaction. Among them, IN W131A and IN Q168L, that were previously identified to be deficient for LEDGF/p75 interaction, were also partially impaired for TNPO3 binding. We observed that mutations abolishing IN ability to form tetramers resulted in a severe reduction in LEDGF/p75 binding. In sharp contrast, no correlation could be found between the ability of IN to multimerize and TNPO3 interaction. Most of the mutant viruses were essentially impaired for the integration step whereas the amount of 2-LTR circles, reflecting the nuclear import of the viral DNA, was not significantly affected. CONCLUSION: Our functional analysis of HIV-1 IN mutants reveals distinct structural basis for TNPO3 interaction and suggests that the interaction between IN and TNPO3 is not a major determinant of nuclear import but could take place at a nuclear step prior to integration.


Asunto(s)
ADN Complementario/metabolismo , Integrasa de VIH/metabolismo , VIH-1/enzimología , Mutación , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virología , ADN Complementario/genética , ADN Viral/genética , ADN Viral/metabolismo , Activación Enzimática , Células HEK293 , Integrasa de VIH/genética , VIH-1/genética , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Transfección , Integración Viral , Replicación Viral , beta Carioferinas/genética
7.
Methods ; 47(4): 291-7, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19232540

RESUMEN

Here we describe methods developed based on systematic yeast two-hybrid screenings that allowed us to identify several binding partners of HIV-1 integrase. We have developed an efficient strategy to perform large comprehensive screenings with different highly complex cDNA libraries derived both random- and oligo-dT primed reactions. A very efficient mating procedure was used for screening in yeast, allowing genetic saturation of positive clones. This importantly leads with confidence to the determination of the regions within the participating proteins responsible for the interactions. Several additional tools were used that allowed us to assess the specificity of the interactions detected, including rebound screens with cellular co-factors as baits performed against a library of random fragments of HIV-1 proviral DNA. For some of the identified cell factors, we have generated and characterized loss of affinity mutants of integrase, which, when combined with viral functional assays, validated the involvement of human lens epithelium-derived growth factor (LEDGF/p75) in the integration step of the HIV-1 replication cycle. All tolled, our studies identified LEDGF/p75, Transportin-SR2 (TNPO3), von Hippel-Lindau binding protein 1 (VBP1), and sucrose non-fermenting 5 (SNF5) as cellular binding partners of HIV-1 integrase.


Asunto(s)
Integrasa de VIH/metabolismo , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/metabolismo , Técnicas del Sistema de Dos Híbridos , Integración Viral/fisiología , Biblioteca de Genes , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos
8.
Cell Rep ; 3(4): 1036-43, 2013 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-23602554

RESUMEN

SAMHD1 restricts HIV-1 replication in myeloid and quiescent CD4(+) T cells. Here, we show that SAMHD1 restriction activity is regulated by phosphorylation. SAMHD1 interacts with cyclin A2/cdk1 only in cycling cells. Cyclin A2/CDK1 phosphorylates SAMHD1 at the Threonine 592 residue both in vitro and in vivo. Phosphorylation of SAMHD1 Thr592 correlates with loss of its ability to restrict HIV-1. Indeed, while PMA treatment of proliferating THP1 cells results in reduced Thr592 phosphorylation, activation of resting peripheral blood mononuclear cells (PBMCs) and purified quiescent CD4(+) T cells results in increased phosphorylation of SAMHD1 Thr592. Interestingly, we found that treatment of cells by type 1 interferon reduced Thr592 phosphorylation, reinforcing the link between the phosphorylation of SAMHD1 and its antiviral activity. Unlike wild-type SAMHD1, a phosphorylation-defective mutant was able to restrict HIV-1 replication in both PMA-treated and untreated cells. Our results uncover the phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 as a key regulatory mechanism of its antiviral activity.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina A2/metabolismo , VIH-1/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Animales , Antivirales/farmacología , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Humanos , Interferón Tipo I/farmacología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD , Alineación de Secuencia , Replicación Viral/efectos de los fármacos
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