RESUMEN
Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria-derived succinate that accumulated both in the respiratory fluids of virus-challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus-triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate-dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Neumonía , Animales , Antivirales/farmacología , Humanos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Ratones , Proteínas de la Nucleocápside , Nucleoproteínas/metabolismo , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Ácido Succínico/uso terapéutico , Replicación ViralRESUMEN
Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies.
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COVID-19 , Citocinas , Pulmón , Macrófagos Alveolares , Macrófagos , SARS-CoV-2 , Humanos , COVID-19/virología , COVID-19/inmunología , SARS-CoV-2/fisiología , Pulmón/virología , Pulmón/inmunología , Pulmón/patología , Macrófagos/virología , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos Alveolares/virología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Citocinas/metabolismo , Monocitos/virología , Monocitos/metabolismo , Monocitos/inmunología , Masculino , Femenino , Análisis de la Célula Individual , Persona de Mediana EdadRESUMEN
The development of safe and effective vaccines in a record time after the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a remarkable achievement, partly based on the experience gained from multiple viral outbreaks in the past decades. However, the Coronavirus Disease 2019 (COVID-19) crisis also revealed weaknesses in the global pandemic response and large gaps that remain in our knowledge of the biology of coronaviruses (CoVs) and influenza viruses, the 2 major respiratory viruses with pandemic potential. Here, we review current knowns and unknowns of influenza viruses and CoVs, and we highlight common research challenges they pose in 3 areas: the mechanisms of viral emergence and adaptation to humans, the physiological and molecular determinants of disease severity, and the development of control strategies. We outline multidisciplinary approaches and technological innovations that need to be harnessed in order to improve preparedeness to the next pandemic.
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COVID-19/virología , Gripe Humana/virología , Orthomyxoviridae/fisiología , SARS-CoV-2/fisiología , Animales , Antivirales , COVID-19/terapia , COVID-19/transmisión , Desarrollo de Medicamentos , Evolución Molecular , Humanos , Gripe Humana/terapia , Gripe Humana/transmisión , Orthomyxoviridae/inmunología , SARS-CoV-2/inmunología , Selección Genética , Carga Viral , Vacunas ViralesRESUMEN
PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus-infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2-induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.
Asunto(s)
Infecciones por Orthomyxoviridae/metabolismo , Multimerización de Proteína , Respiración , Transducción de Señal , Proteínas Virales/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Femenino , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Infecciones por Orthomyxoviridae/fisiopatología , Infecciones por Orthomyxoviridae/virología , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteínas Virales/genéticaRESUMEN
Scour is a hydraulic risk threatening the stability of bridges in fluvial and coastal areas. Therefore, developing permanent and real-time monitoring techniques is crucial. Recent advances in strain measurements using fiber optic sensors allow new opportunities for scour monitoring. In this study, the innovative optical frequency domain reflectometry (OFDR) was used to evaluate the effect of scour by performing distributed strain measurements along a rod under static lateral loads. An analytical analysis based on the Winkler model of the soil was carefully established and used to evaluate the accuracy of the fiber optic sensors and helped interpret the measurements results. Dynamic tests were also performed and results from static and dynamic tests were compared using an equivalent cantilever model.
RESUMEN
PB1-F2 is a virulence factor of influenza A virus (IAV) whose functions remain misunderstood. The different roles of PB1-F2 may be linked to its structural polymorphism and to its propensity to assemble into oligomers and amyloid fibers in the vicinity of the membrane of IAV-infected cells. Here, we monitored the impact of PB1-F2 on the biochemical composition and protein structures of human epithelial pulmonary cells (A549) and monocytic cells (U937) upon IAV infection using synchrotron Fourier-transform infrared (FTIR) and deep UV (DUV) microscopies at the single-cell level. Cells were infected with a wild-type IAV and its PB1-F2 knock-out mutant for analyses at different times post-infection. IR spectra were recorded in each condition and processed to evaluate the change in the component band of the spectra corresponding to the amide I (secondary structure) and the CH stretching region (membrane). The IR spectra analysis revealed that expression of PB1-F2 in U937 cells, but not in A549 cells, results in the presence of a specific ß-aggregate signature. Furthermore, the lipid membrane composition of U937 cells expressing PB1-F2 was also altered in a cell type-dependent manner. Using DUV microscopy and taking advantage of the high content of tryptophan residues in the sequence of PB1-F2 (5/90 aa), we showed that the increase of the autofluorescent signal recorded in monocytic cells could be correlated with the IR detection of ß-aggregates. Altogether, our results constitute an important step forward in the understanding of the cell type-dependent function of PB1-F2.
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Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Agregado de Proteínas , Proteínas Virales/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/virología , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/genética , Microscopía Fluorescente , Células U937 , Proteínas Virales/genéticaRESUMEN
PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.
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Amiloide/toxicidad , Liposomas/metabolismo , Proteínas Virales/toxicidad , Antiinfecciosos/farmacología , Bacillus subtilis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Escherichia coli/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/patogenicidad , Virus de la Influenza A/ultraestructura , Liposomas/ultraestructura , Pruebas de Sensibilidad Microbiana , Permeabilidad , Agregado de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Virales/químicaRESUMEN
PB1-F2 is a viral protein encoded by influenza A viruses (IAVs). PB1-F2 is implicated in virulence by triggering immune cell apoptosis and enhancing inflammation. To obtain an insight into the molecular mechanisms of PB1-F2-mediated virulence, we used the yeast two-hybrid approach to find new PB1-F2 cellular interactors. This allowed us to identify calcium-binding and coiled-coil domain 2 (CALCOCO2, also known as NDP52) as a binding partner of PB1-F2. Binding of PB1-F2 to CALCOCO2 was confirmed by pull-down. Surface plasmon resonance binding experiments enabled us to estimate the dissociation constant (Kd) of the two partners to be around 20 nM. Using bioinformatics tools, we designed a CALCOCO2 interaction map based on previous knowledge and showed a strong connection between this protein and the type I interferon production pathways and the I-κB kinase/NF-κB signalling pathway. NF-κB reporter assays in which CALCOCO2, MAVS and PB1-F2 were co-expressed showed a cooperation of these three proteins to increase the inflammatory response. By contrast, PB1-F2 inhibits the TBK1-dependent activation of an ISRE reporter plasmid. We also demonstrated that the signal transducer TRAF6 is implicated in the enhancement of NF-κB activity mediated by PB1-F2/CALCOCO2 binding. Altogether, this report provides evidence of an interaction link between PB1-F2 and human proteins, and allows a better understanding of the involvement of PB1-F2 in the pathologic process mediated by IAV.
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Interacciones Huésped-Patógeno , Inmunidad Innata , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Biología Computacional , Humanos , Cinética , Unión Proteica , Mapeo de Interacción de Proteínas , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos HíbridosRESUMEN
Highly pathogenic influenza A viruses (IAV) infections represent a serious threat to humans due to their considerable morbidity and mortality capacities. A good understanding of the molecular mechanisms responsible for the acute lung injury observed during this kind of infection is essential to design adapted therapies. In the current study, using an unbiased transcriptomic approach, we compared the host-responses of mice infected with two different subtypes of IAV: H1N1 vs. H5N1. The host-response comparison demonstrated a clear difference between the transcriptomic profiles of H1N1- and H5N1-infected mice despite identical survival kinetics and similar viral replications. The ontological analysis of the two transcriptomes showed two probable causes of death: induction of an immunopathological state of the lung for the H1N1 strain vs. development of respiratory dysfunction in the case of the H5N1 IAV. Finally, a clear signature responsible for lung edema was specifically associated with the H5N1 infection. We propose a potential mechanism of edema development based on predictive bioinformatics tools.
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Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Animales , Análisis por Conglomerados , Epistasis Genética , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Infecciones por Orthomyxoviridae/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Carga ViralRESUMEN
PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain.
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Amiloide/química , Biopolímeros/química , Cardiolipinas/análisis , Membrana Celular/química , Colesterol/química , Virus de la Influenza A/química , Proteínas Virales/química , Pliegue de ProteínaRESUMEN
In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Membrana Mucosa/inmunología , Nanoestructuras , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Vacunas contra la Influenza/inmunología , Ratones , Microscopía Electrónica de Transmisión , Nucleocápside , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga ViralRESUMEN
PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 µM and from 5 nM to 0.5 µM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent.
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Técnicas Electroquímicas , Virus de la Influenza A/fisiología , Proteínas Virales/análisis , Anticuerpos Monoclonales/inmunología , Benzotiazoles , Técnicas Biosensibles , Línea Celular Tumoral , Humanos , Microscopía de Fuerza Atómica , Multimerización de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Tiazoles/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
ZnMgO nanoparticles have shown potential for medical applications as an efficient antibacterial agent. In this work, we investigate the effect of water and two commonly used cell culture media on the physicochemical properties of ZnMgO nanoparticles in correlation with their cytotoxicity. In vacuum, ZnMgO nanopowder consists of MgO (nanocubes) and ZnO (nanotetrapods and nanorods) particles. Upon exposure to water or the Luria-Bertani solution, ZnO characteristic shapes were not observable while MgO nanocubes transformed into octahedral form. In addition, water caused morphological alternations in form of disordered and fragmented structures. This effect was directly reflected in UV/vis absorption properties of ZnMgO, implying that formation of new states within the band gap of ZnO and redistribution of specific sites on MgO surfaces occurs in the presence of water. In mammalian culture cell medium, ZnMgO nanoparticles were shapeless, agglomerated, and coated with surrounding proteins. Serum albumin was found to adsorb as a major but not the only protein. Adsorbed albumin mainly preserved its α-helix secondary structure. Finally, the cytotoxicity of ZnMgO was shown to strongly depend on the environment: in the presence of serum proteins ZnMgO nanopowder was found to be safe for mammalian cells while highly toxic in a serum-free medium or a medium containing only albumin. Our results demonstrate that nanostructured ZnMgO reaches living cells with modified morphology and surface structure when compared to as-synthesized particles kept in vacuum. In addition, its biocompatibility can be modulated by proteins from biological environment.
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Medios de Cultivo/farmacología , Óxido de Magnesio/química , Óxido de Magnesio/toxicidad , Nanopartículas/química , Agua/farmacología , Óxido de Zinc/química , Óxido de Zinc/toxicidad , Adsorción , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Química Física , Medios de Cultivo/química , Perros , Humanos , Células de Riñón Canino Madin Darby , Tamaño de la Partícula , Albúmina Sérica/química , Propiedades de Superficie , Agua/químicaRESUMEN
Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid-protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.
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Amiloide/metabolismo , Liposomas/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Ligadas a GPI , Membrana Dobles de Lípidos/metabolismo , Enfermedades por PriónRESUMEN
Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence.
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Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/inmunología , Transcriptoma , Proteínas Virales/metabolismo , Animales , Apoptosis , Muerte Celular , Quimiotaxis , Femenino , Eliminación de Gen , Regulación Viral de la Expresión Génica , Ingeniería Genética , Interferón beta/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Neutrófilos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/genética , Transcripción Genética , Proteínas Virales/genética , VirulenciaRESUMEN
In this study, we developed a large-scale screening of bacterial strains in order to identify novel candidate probiotics with immunomodulatory properties. For this, 158 strains, including a majority of lactic acid bacteria (LAB), were screened by two different cellular models: tumor necrosis factor alpha (TNF-α)-activated HT-29 cells and peripheral blood mononuclear cells (PBMCs). Different strains responsive to both models (pro- and anti-inflammatory strains) were selected, and their protective effects were tested in vivo in a murine model of influenza virus infection. Daily intragastric administrations during 10 days before and 10 days after viral challenge (100 PFU of influenza virus H1N1 strain A Puerto Rico/8/1934 [A/PR8/34]/mouse) of Lactobacillus plantarum CNRZ1997, one potentially proinflammatory probiotic strain, led to a significant improvement in mouse health by reducing weight loss, alleviating clinical symptoms, and inhibiting significantly virus proliferation in lungs. In conclusion, in this study, we have combined two cellular models to allow the screening of a large number of LAB for their immunomodulatory properties. Moreover, we identified a novel candidate probiotic strain, L. plantarum CNRZ1997, active against influenza virus infection in mice.
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Factores Inmunológicos/administración & dosificación , Lactobacillus plantarum/aislamiento & purificación , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/patogenicidad , Probióticos/administración & dosificación , Animales , Peso Corporal , Células Cultivadas , Modelos Animales de Enfermedad , Lactobacillus plantarum/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Pulmón/virología , Ratones , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/terapiaRESUMEN
Most influenza viruses express the PB1-F2 protein which is regarded as a virulence factor. However, PB1-F2 behaves differently in avian and mammalian hosts, suggesting that this protein may be involved in the species barrier crossings regularly observed in influenza viruses. To better understand the functions associated with this viral protein, we decided to compare the BioID2-derived proximity interactome of a human PB1-F2 from an H3N2 virus with that of an avian PB1-F2 from an H7N1 strain. The results obtained reveal that the two proteins share only a few interactors and thus common functions. The human virus protein is mainly involved in signaling by Rho GTPases while the avian virus protein is mainly involved in ribonucleoprotein complex biogenesis. PB1-F2 H3N2 interactors include several members of the 14-3-3 protein family, a family of regulatory proteins involved in many signaling pathways. We then validated the interaction with 14-3-3 proteins and were able to show that the association of H3N2-PB1-F2 with YWHAH increased the activity of the antiviral sensor MDA5, while H7N1-PB1-F2 had no effect. Collectively, these results show that PB1-F2 can associate with a large range of protein complexes and exert a wide variety of functions. Furthermore, PB1-F2 interactome differs according to the avian or human origin of the protein.
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Proteínas Aviares , Subtipo H7N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Aviar , Animales , Humanos , Proteínas 14-3-3 , Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , MamíferosRESUMEN
In response to the increasing demand for lung transplantation, ex vivo lung perfusion (EVLP) has extended the number of suitable donor lungs by rehabilitating marginal organs. However despite an expanding use in clinical practice, the responses of the different lung cell types to EVLP are not known. In order to advance our mechanistic understanding and establish a refine tool for improvement of EVLP, we conducted a pioneer study involving single cell RNA-seq on human lungs declined for transplantation. Functional enrichment analyses were performed upon integration of data sets generated at 4 h (clinical duration) and 10 h (prolonged duration) from two human lungs processed to EVLP. Pathways related to inflammation were predicted activated in epithelial and blood endothelial cells, in monocyte-derived macrophages and temporally at 4 h in alveolar macrophages. Pathways related to cytoskeleton signaling/organization were predicted reduced in most cell types mainly at 10 h. We identified a division of labor between cell types for the selected expression of cytokine and chemokine genes that varied according to time. Immune cells including CD4+ and CD8+ T cells, NK cells, mast cells and conventional dendritic cells displayed gene expression patterns indicating blunted activation, already at 4 h in several instances and further more at 10 h. Therefore despite inducing inflammatory responses, EVLP appears to dampen the activation of major lung immune cell types, what may be beneficial to the outcome of transplantation. Our results also support that therapeutics approaches aiming at reducing inflammation upon EVLP should target both the alveolar and vascular compartments.
Asunto(s)
Linfocitos T CD8-positivos , Trasplante de Pulmón , Humanos , Perfusión/métodos , Células Endoteliales , Trasplante de Pulmón/métodos , Pulmón/fisiología , InflamaciónRESUMEN
The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-ß gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-ß expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-ß expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-ß expression and found that PB1-F2-mediated IFN-ß induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.
Asunto(s)
Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Interferón beta/biosíntesis , Mucosa Respiratoria/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Apoptosis/inmunología , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Gripe Humana/metabolismo , Interferón beta/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas Virales/genética , Virulencia/genéticaRESUMEN
The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.