RESUMEN
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
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
Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells.
Asunto(s)
Citocinas/metabolismo , Virus de la Influenza B/química , Ubiquitinas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Interferones/farmacología , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-alpha/beta-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B.
Asunto(s)
Citocinas/metabolismo , Virus de la Influenza B/inmunología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Ubiquitinas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Células COS , Núcleo Celular/metabolismo , Núcleo Celular/virología , Chlorocebus aethiops , Citoplasma/metabolismo , Citoplasma/virología , Perros , Células HeLa , Humanos , Virus de la Influenza B/metabolismo , Gripe Humana/inmunología , Gripe Humana/metabolismo , Riñón/citología , Ratones , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Péptidos , Unión Proteica/inmunología , Proteínas de Unión al ARN , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP.
Asunto(s)
Citocinas/metabolismo , Virus de la Influenza B/inmunología , ARN Viral/biosíntesis , Ubiquitinas/metabolismo , Proteínas no Estructurales Virales/genética , Células A549 , Animales , Línea Celular , Embrión de Pollo , Citocinas/genética , Perros , Endopeptidasas/genética , Humanos , Virus de la Influenza B/genética , Células de Riñón Canino Madin Darby , Unión Proteica/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Viral/genética , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterasa , Ubiquitinas/genética , Replicación Viral/genéticaRESUMEN
Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.
Asunto(s)
Muerte Celular , Glicoproteínas/metabolismo , Interacciones Huésped-Patógeno , Virus de la Influenza A/inmunología , ARN Viral/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Línea Celular , Técnicas de Inactivación de Genes , Genómica , Glicoproteínas/deficiencia , Ratones , Ratones Noqueados , Mutación , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARNRESUMEN
Necroptosis has emerged as an important facet of both host defense and inflammatory disease. In this issue, Rodrigue-Gervais et al. (2014) demonstrate a role for the cell death regulator cIAP2 in maintaining lung homeostasis during influenza infection. Loss of cIAP2 promotes necroptosis of lung tissues, leading to host death.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Pulmón/inmunología , Necrosis/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Mucosa Respiratoria/inmunología , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Humanos , Ubiquitina-Proteína LigasasRESUMEN
Programmed cell death is an important facet of host-pathogen interactions. Although apoptosis has long been implicated as the major form of programmed cell death in host defense, the past decade has seen the emergence of other forms of regulated death, including programmed necrosis. While the molecular mechanisms of programmed necrosis continue to be unveiled, an increasing number of viral and bacterial pathogens induce this form of death in host cells, with important consequences for infection, control, and pathogenesis. Moreover, pathogen strategies to manipulate or utilize this pathway are now being discovered. In this review, we focus on a variety of viral and bacterial pathogens where a role for programmed necrosis is starting to be appreciated. In particular, we focus on the mechanistic details of how the host or the pathogen might appropriate this pathway for its own benefit.