RESUMEN
The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.
Asunto(s)
Necroptosis , Ácidos Nucleicos , Animales , Apoptosis/fisiología , Caspasa 8/genética , Caspasa 8/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Interferones/metabolismo , Ratones , Nucleotidiltransferasas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos , Diferenciación Celular , ADN Helicasas , Complejos Multiproteicos , Proteínas Nucleares , Proteínas Proto-Oncogénicas c-myc , Factores de Transcripción , Animales , Linfocitos T CD8-positivos/citología , ADN Helicasas/metabolismo , Modelos Animales de Enfermedad , Memoria Inmunológica , Inmunoterapia , Células T de Memoria/citología , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Neoplasias , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Quiméricos de Antígenos , Factores de Transcripción/metabolismoRESUMEN
Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.
Asunto(s)
Linfocitos T CD8-positivos , Factor 4F Eucariótico de Iniciación , Diferenciación Celular , Activación de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina/genéticaRESUMEN
Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Eosinófilos/metabolismo , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Virus de la Influenza A/genética , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN-antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN-suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN-antagonistic properties. Mutation of this position leads to enhanced activation of the IFN-mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.
Asunto(s)
Virus de la Influenza B , Interferón Tipo I/antagonistas & inhibidores , Proteínas de la Nucleocápside/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Proteínas Virales/inmunología , Células A549 , Animales , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Virus de la Influenza B/enzimología , Virus de la Influenza B/inmunología , Gripe Humana/virología , Células VeroRESUMEN
Eosinophils are multifunctional cells of the innate immune system linked to allergic inflammation. Asthmatics were more likely to be hospitalized but less likely to suffer severe morbidity and mortality during the 2009 influenza pandemic. These epidemiologic findings were recapitulated in a mouse model of fungal asthma wherein infection during heightened allergic inflammation was protective against influenza A virus (IAV) infection and disease. Our goal was to delineate a mechanism(s) by which allergic asthma may alleviate influenza disease outcome, focused on the hypothesis that pulmonary eosinophilia linked with allergic respiratory disease is able to promote antiviral host defenses against the influenza virus. The transfer of eosinophils from the lungs of allergen-sensitized and challenged mice into influenza virus-infected mice resulted in reduced morbidity and viral burden, improved lung compliance, and increased CD8+ T cell numbers in the airways. In vitro assays with primary or bone marrow-derived eosinophils were used to determine eosinophil responses to the virus using the laboratory strain (A/PR/08/1934) or the pandemic strain (A/CA/04/2009) of IAV. Eosinophils were susceptible to IAV infection and responded by activation, piecemeal degranulation, and upregulation of Ag presentation markers. Virus- or viral peptide-exposed eosinophils induced CD8+ T cell proliferation, activation, and effector functions. Our data suggest that eosinophils promote host cellular immunity to reduce influenza virus replication in lungs, thereby providing a novel mechanism by which hosts with allergic asthma may be protected from influenza morbidity.
Asunto(s)
Asma/inmunología , Eosinófilos/inmunología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Asma/complicaciones , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Hipersensibilidad/complicaciones , Hipersensibilidad/inmunología , Activación de Linfocitos/inmunología , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Infecciones por Orthomyxoviridae/complicaciones , Eosinofilia Pulmonar/inmunologíaRESUMEN
T cell activation results in a rapidly proliferating T cell endowed with a metabolic phenotype necessary for growth and division. However, before the cell can proceed towards this burst of cell division a phase of quiescence occurs, during which the basic mechanisms governing regulation of metabolic reprograming are established. This review focuses on key cellular processes controlling early metabolic regulation and how these circuits of metabolic control dictate distinct cellular fates upon the first asymmetric division.
Asunto(s)
División Celular Asimétrica , Metabolismo Energético , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , División Celular Asimétrica/genética , División Celular Asimétrica/inmunología , Biomarcadores , Regulación de la Expresión Génica , Humanos , Activación de Linfocitos/genética , Transducción de SeñalRESUMEN
Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.
Asunto(s)
Apoptosis , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/inmunología , Necrosis , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/fisiología , Células Epiteliales/virología , Proteína de Dominio de Muerte Asociada a Fas/genética , Fibroblastos/fisiología , Fibroblastos/virología , Humanos , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/patología , Proteínas Quinasas/genética , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , División Celular , Polaridad Celular , Activación de Linfocitos , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Animales , Diferenciación Celular/genética , Polaridad Celular/genética , Femenino , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Transcripción GenéticaRESUMEN
Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Interferón beta/metabolismo , Treonina/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Proteína 58 DEAD Box/metabolismo , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Interferón beta/genética , Células de Riñón Canino Madin Darby/virología , Mutación , Fosforilación , Regiones Promotoras Genéticas , Receptores Inmunológicos , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Incursions of new pathogenic viruses into humans from animal reservoirs are occurring with alarming frequency. The molecular underpinnings of immune recognition, host responses, and pathogenesis in this setting are poorly understood. We studied pandemic influenza viruses to determine the mechanism by which increasing glycosylation during evolution of surface proteins facilitates diminished pathogenicity in adapted viruses. ER stress during infection with poorly glycosylated pandemic strains activated the unfolded protein response, leading to inflammation, acute lung injury, and mortality. Seasonal strains or viruses engineered to mimic adapted viruses displaying excess glycans on the hemagglutinin did not cause ER stress, allowing preservation of the lungs and survival. We propose that ER stress resulting from recognition of non-adapted viruses is utilized to discriminate "non-self" at the level of protein processing and to activate immune responses, with unintended consequences on pathogenesis. Understanding this mechanism should improve strategies for treating acute lung injury from zoonotic viral infections.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/virología , Estrés del Retículo Endoplásmico/inmunología , Lesión Pulmonar Aguda/metabolismo , Animales , Perros , Glicosilación , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/metabolismo , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Transducción de SeñalRESUMEN
BACKGROUND: Nonstructural protein 1 (NS1) proteins from avian influenza viruses like the 1918 pandemic NS1 are capable of inhibiting the key signaling integrator c-Abl (Abl1), resulting in massive cytopathic cell alterations. METHODS: In the current study, we addressed the consequences of NS1-mediated alteration of c-Abl on acute lung injury and pathogenicity in an in vivo mouse model. RESULTS: Comparing isogenic strains that differ only in their ability to inhibit c-Abl, we observed elevated pathogenicity for the c-Abl-inhibiting virus. NS1-mediated blockade of c-Abl resulted in severe lung pathology and massive edema formation and facilitated secondary bacterial pneumonia. This phenotype was independent of differences in replication and immune responses, defining it as an NS1 virulence mechanism distinct from its canonical functions. Microarray analysis revealed extensive downregulation of genes involved in cell integrity and vascular endothelial regulation. CONCLUSIONS: NS1 protein-mediated blockade of c-Abl signaling drives acute lung injury and primes for bacterial coinfections revealing potential insights into the pathogenicity of the 1918 pandemic virus.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Infecciones Bacterianas/etiología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Influenza Pandémica, 1918-1919 , Infecciones por Orthomyxoviridae/virología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas no Estructurales Virales/metabolismo , Lesión Pulmonar Aguda/complicaciones , Lesión Pulmonar Aguda/patología , Animales , Biomarcadores , Humanos , Pulmón/patología , Pulmón/virología , Ratones , Proteínas Proto-Oncogénicas c-abl/genéticaRESUMEN
The type I interferon (IFN) response represents the first line of defence to invading pathogens. Internalized viral ribonucleoproteins (vRNPs) of negative-strand RNA viruses induce an early IFN response by interacting with retinoic acid inducible gene I (RIG-I) and its recruitment to mitochondria. Here we employ three-dimensional stochastic optical reconstruction microscopy (STORM) to visualize incoming influenza A virus (IAV) vRNPs as helical-like structures associated with mitochondria. Unexpectedly, an early IFN induction in response to vRNPs is not detected. A distinct amino-acid motif in the viral polymerases, PB1/PA, suppresses early IFN induction. Mutation of this motif leads to reduced pathogenicity in vivo, whereas restoration increases it. Evolutionary dynamics in these sequences suggest that completion of the motif, combined with viral reassortment can contribute to pandemic risks. In summary, inhibition of the immediate anti-viral response is 'pre-packaged' in IAV in the sequences of vRNP-associated polymerase proteins.
Asunto(s)
ARN Helicasas DEAD-box/química , Virus de la Influenza A/patogenicidad , Interferón Tipo I/inmunología , Mitocondrias/virología , Virión/química , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Proteína 58 DEAD Box , Evolución Molecular , Femenino , Humanos , Imagenología Tridimensional , Pulmón/inmunología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Virus ARN/patogenicidad , Receptores Inmunológicos , Carga Viral , Proteínas Virales/química , VirulenciaRESUMEN
The non-structural protein 1 (NS1) of influenza A viruses (IAV) encodes several src homology (SH) binding motifs (bm) (one SH2bm, up to two SH3bm), which mediate interactions with host cell proteins. In contrast to NS1 of human IAV, NS1 of avian strains possess the second SH3bm (SH3(II)bm) consensus sequence. Since our former studies demonstrated an NS1-CRK interaction, mediated by this motif, here, we addressed the regulatory properties of this SH3bm for cellular signalling. Initially, we observed a reduced basal CRK phosphorylation upon infection with avian IAV harbouring an NS1 with an SH3(II)bm in contrast to human IAV. Reduced activity of the tyrosine kinase c-Abl was identified to be responsible for reduced CRK phosphorylation. Further, binding of NS1 to c-Abl was determined, and mutational manipulation of the SH3(II)bm illustrated the necessity of this motif for c-Abl inhibition. Interestingly, Abl kinase inhibition resulted in impaired avian IAV propagation and pathogenicity and mutational analysis linked the pronounced inhibition of c-Abl to cytopathogenic cell alterations upon avian IAV infections. Taken together, NS1 proteins of avian IAV interfere with the kinase activity of c-Abl, a major cellular signalling integrator that controls multiple signalling processes and cell fate regulations apparently including IAV infections.
Asunto(s)
Interacciones Huésped-Patógeno , Virus de la Influenza A/fisiología , Gripe Aviar/virología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Aves , Línea Celular , Humanos , Virus de la Influenza A/aislamiento & purificación , Unión ProteicaRESUMEN
The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu. To circumvent the development of resistance, recent antiviral approaches target cellular proteins needed by the virus for efficient replication. We investigated the contribution of the small GTPase Rac1 to the replication of influenza viruses. Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains. Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex. The antiviral potential of NSC23766 was confirmed in mouse experiments, identifying Rac1 as a new cellular target for therapeutic treatment of influenza virus infections.
Asunto(s)
Aminoquinolinas/química , Antivirales/química , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Orthomyxoviridae/enzimología , Pirimidinas/química , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Apoptosis , Línea Celular Tumoral , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Polimerasa Dependiente del ARN/metabolismo , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
The identification of amino acid motifs responsible for increased virulence and/or transmission of influenza viruses is of enormous importance to predict pathogenicity of upcoming influenza strains. We phenotypically and genotypically compared 2 variants of influenza virus A/PR/8/34 with different passage histories. The analysis revealed differences in virulence due to an altered type I interferon (IFN) induction, as evidenced by experiments using IFNAR(-/-) mice. Interestingly, these differences were not due to altered functions of the well-known viral IFN antagonists NS1 or PB1-F2. Using reassortant viruses, we showed that differences in the polymerase proteins and nucleoprotein determined the altered virulence. In particular, changes in PB1 and PA contributed to an altered host type I IFN response, indicating IFN antagonistic properties of these proteins. Thus, PB1 and PA appear to harbor previously unknown virulence markers, which may prove helpful in assessing the risk potential of emerging influenza viruses.