RESUMO
The search for new active substances against SARS-CoV-2 is still a central challenge after the COVID-19 pandemic. Antiviral agents to complement vaccination are an important pillar in the clinical situation. Selected cannabinoids such as cannabigerol, cannabicyclol, cannabichromene, and cannabicitran from Cannabis sativa and synthetic homologues of cannabigerol and cannabicyclol were evaluated for effects on the cell viability of Vero cells (CC50 of cannabigerol and cannabicyclol 40 resp. 38 µM) and reduced virus entry of vesicular stomatitis pseudotyped viruses with surface-expressed SARS-CoV-2 spike protein at 20 µM. In addition to a reduction of pseudotyped virus entry, a titer reduction assay on Vero cells after preincubation of Wuhan SARS-CoV-2 significantly confirmed antiviral activity. Investigations on the molecular targets addressed by cannabigerol and cannabicyclol indicated that both compounds are inhibitors of SARS-CoV-2 spike protein-mediated membrane fusion, as could be shown by a virus-free reporter fusion inhibition assay (EC50 for cannabigerol 5.5 µM and for cannabicyclol 10.8 µM) and by monitoring syncytia formation in Vero reporter cells. Selectivity indices were calculated as 7.4 for cannabigerol and 3.5 for cannabicyclol. Systematic semisynthetic alterations of cannabigerol and cannabicyclol indicated that the side chains of both compounds do not contribute to the observed anti-membrane fusion activity.
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Antivirais , Canabinoides , SARS-CoV-2 , Internalização do Vírus , Chlorocebus aethiops , Células Vero , Animais , SARS-CoV-2/efeitos dos fármacos , Canabinoides/farmacologia , Antivirais/farmacologia , Internalização do Vírus/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Humanos , Sobrevivência Celular/efeitos dos fármacos , Tratamento Farmacológico da COVID-19 , Cannabis/químicaRESUMO
Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.
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Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.
Assuntos
Vírus da Influenza A , Vírus da Influenza A/genética , Replicação Viral/fisiologia , Transcrição Gênica , Nucleotidiltransferases/metabolismo , Genômica , Glicólise , RNA Viral/genética , RNA Viral/metabolismoRESUMO
For almost two years, the COVID-19 pandemic has constituted a major challenge to human health, particularly due to the lack of efficient antivirals to be used against the virus during routine treatment interventions. Multiple treatment options have been investigated for their potential inhibitory effect on SARS-CoV-2. Natural products, such as plant extracts, may be a promising option, as they have shown an antiviral activity against other viruses in the past. Here, a quantified extract of Hypericum perforatum was tested and found to possess a potent antiviral activity against SARS-CoV-2. The antiviral potency of the extract could be attributed to the naphtodianthrones hypericin and pseudohypericin, in contrast to other tested ingredients of the plant material, which did not show any antiviral activity. Hypericum perforatum and its main active ingredient hypericin were also effective against different SARS-CoV-2 variants (Alpha, Beta, Delta, and Omicron). Concerning its mechanism of action, evidence was obtained that Hypericum perforatum and hypericin may hold a direct virus-blocking effect against SARS-CoV-2 virus particles. Taken together, the presented data clearly emphasize the promising antiviral activity of Hypericum perforatum and its active ingredients against SARS-CoV-2 infections.
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BACKGROUND: During shortages of filtering face pieces (FFP) in a pandemic, it is necessary to implement a method for safe reuse or extended use. Our aim was to develop a simple, inexpensive and ecological method for decontamination of disposable FFPs that preserves filtration efficiency and material integrity. MATERIAL AND METHODS: Contamination of FFPs (3M Aura 9320+) with SARS-CoV-2 (1.15 × 104 PFUs), Enterococcus faecium (>106 CFUs), and physiological nasopharyngeal flora was performed prior to decontamination by submersion in a solution of 6 % acetic acid and 6 % hydrogen peroxide (6%AA/6%HP solution) over 30 minutes. Material integrity was assessed by testing the filtering efficiency, loss of fit and employing electron microscopy. RESULTS AND DISCUSSION: Decontamination with the 6%AA/6%HP solution resulted in the complete elimination of SARS-CoV-2, E. faecium and physiological nasopharyngeal flora. Material characterization post-treatment showed neither critical material degradation, loss of fit or reduction of filtration efficiency. Electron microscopy revealed no damage to the fibers, and the rubber bands' elasticity was not affected by the decontamination procedure. No concerning residuals of the decontamination procedure were found. CONCLUSION: The simple application and widespread availability of 6%AA/6%HP solution for decontaminating disposable FFPs make this solution globally viable, including developing and third world countries.
Assuntos
COVID-19 , Pandemias , COVID-19/prevenção & controle , Descontaminação/métodos , Reutilização de Equipamento , Humanos , Pandemias/prevenção & controle , Ácido Peracético/farmacologia , SARS-CoV-2 , Ventiladores MecânicosRESUMO
Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.
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Ciclo do Ácido Cítrico/fisiologia , Metabolismo Energético/fisiologia , Ácidos Graxos/biossíntese , Glicólise/fisiologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Adenoviridae/metabolismo , Coronavirus/metabolismo , Humanos , Orthomyxoviridae/metabolismo , Vírus da Parainfluenza 1 Humana/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Rhinovirus/metabolismoRESUMO
BACKGROUND: Secondary bacterial coinfections are ranked as a leading cause of hospitalization and morbid conditions associated with influenza. Because vitamin A deficiency (VAD) and insufficiency are frequent in both developed and developing countries, we asked how VAD influences coinfection severity. METHODS: VAD and control mice were infected with influenza virus for evaluation of inflammatory cytokines, cellular immune responses, and viral clearance. Influenza-infected mice were coinfected with Streptococcus pneumoniae to study weight loss and survival. RESULTS: Naive VAD mouse lungs exhibited dysregulated immune function. Neutrophils were enhanced in frequency and there was a significant reduction in RANTES (regulated on activation of normal T cells expressed and secreted), a chemokine instrumental in T-cell homing and recruitment. After influenza virus infection, VAD mice experienced failures in CD4+ T-cell recruitment and B-cell organization into lymphoid structures in the lung. VAD mice exhibited higher viral titers than controls and slow viral clearance. There were elevated levels of inflammatory cytokines and innate cell subsets in the lungs. However, arginase, a marker of alternatively activated M2 macrophages, was rare. When influenza-infected VAD animals were exposed to bacteria, they experienced a 100% mortality rate. CONCLUSION: Data showed that VAD dysregulated the immune response. Consequently, secondary bacterial infections were 100% lethal in influenza-infected VAD mice.
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Coinfecção , Infecções por Orthomyxoviridae , Infecções Pneumocócicas/complicações , Deficiência de Vitamina A , Animais , Citocinas , Imunidade , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae , Infecções por Orthomyxoviridae/complicações , Infecções Pneumocócicas/mortalidade , Streptococcus pneumoniae , Deficiência de Vitamina A/complicaçõesRESUMO
Cells respond to extracellular agents by activation of intracellular signaling pathways. Viruses can be regarded as such agents, leading to a firework of signaling inside the cell, primarily induced by pathogen-associated molecular patterns (PAMPs) that provoke safeguard mechanisms to defend from the invader. In the constant arms race between pathogen and cellular defense, viruses not only have evolved mechanisms to suppress or misuse supposedly antiviral signaling processes for their own benefit but also actively induce signaling to promote replication. This creates viral dependencies that may be exploited for novel strategies of antiviral intervention. Here, we will summarize the current knowledge of activation and function of influenza virus-induced signaling pathways with a focus on nuclear factor (NF)-κB signaling, mitogen-activated protein kinase cascades, and the phosphatidylinositol-3-kinase pathway. We will discuss the opportunities and drawbacks of targeting these signaling pathways for antiviral intervention.
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Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , NF-kappa B/fisiologia , Orthomyxoviridae/crescimento & desenvolvimento , Proteínas não Estruturais Virais/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world's population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.
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Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection.
Assuntos
Antivirais/efeitos adversos , Aspirina/análogos & derivados , Infecções Bacterianas/etiologia , Glicina/efeitos adversos , Influenza Humana/tratamento farmacológico , Lisina/análogos & derivados , NF-kappa B/antagonistas & inibidores , Células A549 , Aspirina/efeitos adversos , Combinação de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Técnicas de Silenciamento de Genes , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/complicações , Influenza Humana/virologia , Lisina/efeitos adversos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/etiologia , Superinfecção/etiologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Replicação Viral/efeitos dos fármacosRESUMO
Activation of the Raf/MEK/ERK cascade is required for efficient propagation of several RNA and DNA viruses, including human respiratory syncytial virus (RSV). In RSV infection, activation of the Raf/MEK/ERK cascade is biphasic. An early induction within minutes after infection is associated with viral attachment. Subsequently, a second activation occurs with, so far, unknown function in the viral life cycle. In this study, we aimed to characterise the role of Raf/MEK/ERK-mediated signalling during ongoing RSV infection. Our data show that inhibition of the kinase MEK after the virus has been internalised results in a reduction of viral titers. Further functional investigations revealed that the late-stage activation of ERK is required for a specific step in RSV replication, namely, the secretory transport of the RSV fusion protein F. Thus, MEK inhibition resulted in impaired surface accumulation of the F protein. F protein surface expression is essential for efficient replication as it is involved in viral filament formation, cell fusion, and viral transmission. In summary, we provide detailed insights of how host cell signalling interferes with RSV replication and identified the Raf/MEK/ERK kinase cascade as potential target for novel anti-RSV strategies.
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Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Sistema de Sinalização das MAP Quinases , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais de Fusão/metabolismo , Replicação Viral , Animais , Células Cultivadas , Humanos , Transporte ProteicoRESUMO
The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2-/- knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2-/- as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2-/- knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2-/- knock-out mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.
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Hemaglutininas/metabolismo , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/virologia , Serina Endopeptidases/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Clonagem Molecular , Cães , Regulação Viral da Expressão Gênica/fisiologia , Hemaglutininas/química , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutagênese , Conformação Proteica , Serina Endopeptidases/genéticaRESUMO
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.
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Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/virologia , Estresse do Retículo Endoplasmático/imunologia , Lesão Pulmonar Aguda/metabolismo , Animais , Cães , Glicosilação , Células HEK293 , Humanos , Imunidade Inata/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Transdução de SinaisRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/etiologia , Infecções Bacterianas/etiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Pandêmica, 1918-1919 , Infecções por Orthomyxoviridae/virologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas não Estruturais Virais/metabolismo , Lesão Pulmonar Aguda/complicações , Lesão Pulmonar Aguda/patologia , Animais , Biomarcadores , Humanos , Pulmão/patologia , Pulmão/virologia , Camundongos , Proteínas Proto-Oncogênicas c-abl/genéticaRESUMO
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.
Assuntos
RNA Helicases DEAD-box/química , Vírus da Influenza A/patogenicidade , Interferon Tipo I/imunologia , Mitocôndrias/virologia , Vírion/química , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Proteína DEAD-box 58 , Evolução Molecular , Feminino , Humanos , Imageamento Tridimensional , Pulmão/imunologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Vírus de RNA/patogenicidade , Receptores Imunológicos , Carga Viral , Proteínas Virais/química , VirulênciaRESUMO
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.
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Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Animais , Aves , Linhagem Celular , Humanos , Vírus da Influenza A/isolamento & purificação , Ligação ProteicaRESUMO
UNLABELLED: Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE: Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.
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Antivirais/metabolismo , Vírus da Influenza A/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Cães , Células HEK293 , Humanos , Vírus da Influenza A/genética , Interferon beta/genética , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Regiões Promotoras Genéticas/genética , Proteômica/métodos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Receptores Imunológicos , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
UNLABELLED: A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE: Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection.
Assuntos
Sequência de Aminoácidos/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Infecções por Orthomyxoviridae/genética , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Cães , Células HEK293 , Humanos , Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Influenza Humana/virologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida/métodos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Transdução de Sinais/genética , Tretinoína/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
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.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Galinhas , Citocinas/análise , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Interferon Tipo I/metabolismo , Estimativa de Kaplan-Meier , Pulmão/química , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1 , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Virulência/imunologiaRESUMO
The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.