RESUMEN
Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.
Asunto(s)
Cardiotoxicidad , ADN Mitocondrial , Animales , Ratones , ADN Mitocondrial/metabolismo , Inmunidad Innata , Interferones/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , FosforilaciónRESUMEN
Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.
Asunto(s)
Virus de la Influenza A/genética , Necroptosis/genética , Proteínas de Unión al ARN/metabolismo , Animales , Apoptosis/genética , Muerte Celular/genética , Línea Celular Tumoral , Femenino , Virus de la Influenza A/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , ARN/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiologíaRESUMEN
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.
Asunto(s)
Muerte Celular Inmunogénica , Proteolisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Transducción de Señal , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Humanos , Animales , Ratones , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de los fármacos , Necroptosis/efectos de los fármacos , Necroptosis/inmunología , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Ratones Endogámicos C57BL , Antineoplásicos/farmacología , Inmunoterapia/métodosRESUMEN
Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the ß-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Muromegalovirus/metabolismo , Necroptosis/fisiología , Neuropilina-1/metabolismo , Animales , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Infecciones por Citomegalovirus/genética , Eliminación de Gen , Regulación Viral de la Expresión Génica , Ratones , Muromegalovirus/genética , Neuropilina-1/genéticaRESUMEN
Programmed cell death is an integral part of host defense against invading intracellular pathogens. Apoptosis, programmed necrosis, and pyroptosis each serve to limit pathogen replication in infected cells, while simultaneously promoting the inflammatory and innate responses that shape effective long-term host immunity. The importance of carefully regulated cell death is evident in the spectrum of inflammatory and autoimmune disorders caused by defects in these pathways. Moreover, many viruses encode inhibitors of programmed cell death to subvert these host responses during infection, thereby facilitating their own replication and persistence. Thus, as both virus and cell vie for control of these pathways, the battle for survival has shaped a complex host-pathogen interaction. This review will discuss the multifaceted role that programmed cell death plays in maintaining the immune system and its critical function in host defense, with a special emphasis on viral infections.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Mamíferos/virología , Modelos Inmunológicos , Virosis/inmunología , Animales , Muerte Celular/inmunología , Linfocitos/inmunología , Linfocitos/virologíaRESUMEN
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.
Asunto(s)
Apoptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Animales , Caspasa 8/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Técnicas de Sustitución del Gen , Células HT29 , Humanos , Ratones , Ratones Transgénicos , Células 3T3 NIH , Necrosis/enzimología , Proteínas de Complejo Poro Nuclear/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidoresRESUMEN
Nucleic acids are potent triggers for innate immunity. Double-stranded DNA and RNA adopt different helical conformations, including the unusual Z-conformation. Z-DNA/RNA is recognised by Z-binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM-1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z-DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1-ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA These observations show that Z-RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas/metabolismo , ARN/metabolismo , Animales , Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Glicoproteínas/genética , Ratones , Ratones Transgénicos , Células 3T3 NIH , Conformación de Ácido Nucleico , ARN/química , Proteínas de Unión al ARNRESUMEN
The programmed self-destruction of infected cells is a powerful antimicrobial strategy in metazoans. For decades, apoptosis represented the dominant mechanism by which the virus-infected cell was thought to undergo programmed cell death. More recently, however, new mechanisms of cell death have been described that are also key to host defense. One such mechanism in vertebrates is programmed necrosis, or "necroptosis", driven by receptor-interacting protein kinase 3 (RIPK3). Once activated by innate immune stimuli, including virus infections, RIPK3 phosphorylates the mixed lineage kinase domain-like protein (MLKL), which then disrupts cellular membranes to effect necroptosis. Emerging evidence demonstrates that RIPK3 can also mediate apoptosis and regulate inflammasomes. Here, we review studies on the mechanisms by which viruses activate RIPK3 and the pathways engaged by RIPK3 that drive cell death.
Asunto(s)
Inflamasomas/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Virosis/inmunología , Virus/inmunología , Animales , Apoptosis , Humanos , Inmunidad Innata , NecrosisRESUMEN
Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.
Asunto(s)
ADN de Forma Z/metabolismo , Glicoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Virus Vaccinia/metabolismo , Proteínas Virales/metabolismo , Animales , Caspasas/metabolismo , Muerte Celular , Línea Celular , Supervivencia Celular , ADN de Forma Z/química , Glicoproteínas/genética , Humanos , Inmunidad Innata , Interferón Tipo I/química , Interferón Tipo I/farmacología , Ratones , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Proteínas Virales/química , VirulenciaRESUMEN
DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) is a crucial sensor of necroptotic cell death induced by murine cytomegalovirus (MCMV) in its natural host. Here, we show that viral capsid transport to the nucleus and subsequent viral IE3-dependent early transcription are required for necroptosis. Necroptosis induction does not depend on input virion DNA or newly synthesized viral DNA A putative RNA-binding domain of DAI/ZBP1, Zα2, is required to sense virus and trigger necroptosis. Thus, MCMV IE3-dependent transcription from the viral genome plays a crucial role in activating DAI/ZBP1-dependent necroptosis. This implicates RNA transcripts generated by a large double-stranded DNA virus as a biologically relevant ligand for DAI/ZBP1 during natural viral infection.
Asunto(s)
Apoptosis , Glicoproteínas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Muromegalovirus/fisiología , Necrosis , Transcripción Genética , Animales , Muerte Celular , Proteínas de Unión al ADN/metabolismo , Glicoproteínas/genética , Proteínas Inmediatas-Precoces/genética , Ratones , Muromegalovirus/genética , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
We report the specific collision of a single murine cytomegalovirus (MCMV) on a platinum ultramicroelectrode (UME, radius of 1 µm). Antibody directed against the viral surface protein glycoprotein B functionalized with glucose oxidase (GOx) allowed for specific detection of the virus in solution and a biological sample (urine). The oxidation of ferrocene methanol to ferrocenium methanol was carried out at the electrode surface, and the ferrocenium methanol acted as the cosubstrate to GOx to catalyze the oxidation of glucose to gluconolactone. In the presence of glucose, the incident collision of a GOx-covered virus onto the UME while ferrocene methanol was being oxidized produced stepwise increases in current as observed by amperometry. These current increases were observed due to the feedback loop of ferrocene methanol to the surface of the electrode after GOx reduces ferrocenium methanol back to ferrocene. Negative controls (i) without glucose, (ii) with an irrelevant virus (murine gammaherpesvirus 68), and (iii) without either virus do not display these current increases. Stepwise current decreases were observed for the prior two negative controls and no discrete events were observed for the latter. We further apply this method to the detection of MCMV in urine of infected mice. The method provides for a selective, rapid, and sensitive detection technique based on electrochemical collisions.
Asunto(s)
Citomegalovirus , Técnicas Electroquímicas , Compuestos Ferrosos/química , Glucosa Oxidasa/química , Glucosa/química , Orina/virología , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Ratones Endogámicos C57BL , Microelectrodos , Platino (Metal)/química , Proteínas del Envoltorio Viral/inmunología , ViriónRESUMEN
Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus.IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.
Asunto(s)
Muerte Celular , Interacciones Huésped-Patógeno , Inmunidad Innata , ARN Viral/metabolismo , Reoviridae/inmunología , Reoviridae/fisiología , Animales , Línea Celular , Fibroblastos/inmunología , Fibroblastos/fisiología , Fibroblastos/virología , RatonesRESUMEN
We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead "anchors," which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes.
Asunto(s)
Anticuerpos Antivirales/química , Citomegalovirus/química , Técnicas Electroquímicas/métodos , Animales , Humanos , Ratones , Microelectrodos , Células 3T3 NIHRESUMEN
Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and ß-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.
Asunto(s)
ADN Viral/inmunología , Gammaherpesvirinae/inmunología , Infecciones por Herpesviridae/inmunología , Inmunidad Innata/inmunología , Latencia del Virus/inmunología , Animales , Citosol/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.
Asunto(s)
Apoptosis , Caspasa 8/genética , Caspasa 8/metabolismo , Pérdida del Embrión/genética , Pérdida del Embrión/metabolismo , Eliminación de Gen , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Inhibidores de Caspasas , Línea Celular , Pérdida del Embrión/enzimología , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Inmunocompetencia/genética , Inmunocompetencia/inmunología , Enfermedades Linfáticas/genética , Enfermedades Linfáticas/inmunología , Enfermedades Linfáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Programmed necrosis mediated by receptor interacting protein kinase (RIP)3 (also called RIPK3) has emerged as an alternate death pathway triggered by TNF family death receptors, pathogen sensors, IFNRs, Ag-specific TCR activation, and genotoxic stress. Necrosis leads to cell leakage and acts as a "trap door," eliminating cells that cannot die by apoptosis because of the elaboration of pathogen-encoded caspase inhibitors. Necrotic signaling requires RIP3 binding to one of three partners-RIP1, DAI, or TRIF-via a common RIP homotypic interaction motif. Once activated, RIP3 kinase targets the pseudokinase mixed lineage kinase domain-like to drive cell lysis. Although necrotic and apoptotic death can enhance T cell cross-priming during infection, mice that lack these extrinsic programmed cell death pathways are able to produce Ag-specific T cells and control viral infection. The entwined relationship of apoptosis and necrosis evolved in response to pathogen-encoded suppressors to support host defense and contribute to inflammation.
Asunto(s)
Proteínas de la Cápside/inmunología , Tolerancia Inmunológica , Linfocitos T/inmunología , Virosis/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Secuencias de Aminoácidos , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas de la Cápside/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Ratones , Necrosis/genética , Necrosis/inmunología , Necrosis/patología , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Linfocitos T/patología , Virosis/genética , Virosis/patologíaRESUMEN
Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-ß (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Glicoproteínas/genética , Glicoproteínas/metabolismo , Ratones , Ratones Noqueados , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células 3T3 NIH , Necrosis/genética , Necrosis/metabolismo , Necrosis/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptor Toll-Like 3/genéticaRESUMEN
Cytomegalovirus (CMV) is a widely prevalent herpesvirus that reaches seroprevalence rates of up to 95% in several parts of the world. The majority of CMV infections are asymptomatic, albeit they have severe detrimental effects on immunocompromised individuals. Congenital CMV infection is a leading cause of developmental abnormalities in the USA. CMV infection is a significant risk factor for cardiovascular diseases in individuals of all ages. Like other herpesviruses, CMV regulates cell death for its replication and establishes and maintains a latent state in the host. Although CMV-mediated regulation of cell death is reported by several groups, it is unknown how CMV infection affects necroptosis and apoptosis in cardiac cells. Here, we infected primary cardiomyocytes, the contractile cells in the heart, and primary cardiac fibroblasts with wild-type and cell-death suppressor deficient mutant CMVs to determine how CMV regulates necroptosis and apoptosis in cardiac cells. Our results reveal that CMV infection prevents TNF-induced necroptosis in cardiomyocytes; however, the opposite phenotype is observed in cardiac fibroblasts. CMV infection also suppresses inflammation, reactive oxygen species (ROS) generation, and apoptosis in cardiomyocytes. Furthermore, CMV infection improves mitochondrial biogenesis and viability in cardiomyocytes. We conclude that CMV infection differentially affects the viability of cardiac cells.
RESUMEN
Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.
Asunto(s)
Necroptosis/fisiología , Proteínas de Unión al ARN/metabolismo , Virus Vaccinia/fisiología , Proteínas Virales/metabolismo , Adenosina Desaminasa/metabolismo , Muerte Celular , Humanos , Necroptosis/genética , Proteínas Quinasas/metabolismo , ARN Bicatenario , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Virus Vaccinia/genéticaRESUMEN
Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.