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1. The endocytic recycling compartment serves as a viral factory for hepatitis E virus

Author: Cyrine Bentaleb, Kévin Hervouet, Claire Montpellier, Charline Camuzet, Martin Ferrié, Julien Burlaud-Gaillard, Stéphane Bressanelli, Karoline Metzger, Elisabeth Werkmeister, Maliki Ankavay, Nancy Leon Janampa, Julien Marlet, Julien Roux, Clarence Deffaud, Anne Goffard, Yves Rouillé, Jean Dubuisson, Philippe Roingeard, Cécile-Marie Aliouat-Denis, Laurence Cocquerel, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], Morphogénèse et antigénicité du VIH et du virus des Hépatites [MAVIVH - U1259 Inserm - CHRU Tours ], Institut de Biologie Intégrative de la Cellule [I2BC], Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 [PLBS], BIOTEM [Apprieu], Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Institute of Microbiology [University of Lausanne] (IMUL), Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), and This work has been funded by Inserm-Transfert (MAT-PI-17006), Pasteur Institute of Lille (DiagHepE), Region Hauts-de-France (DiagHepE), ANRS and the University of Tours.
Subjects: medicine.drug_class, viruses, Viral factories, [SDV]Life Sciences [q-bio], Endocytic recycling, RNA-dependent RNA polymerase, Infectious particles, Biology, medicine.disease_cause, Monoclonal antibody, Virus, Antibodies, Cellular and Molecular Neuroscience, 03 medical and health sciences, Hepatitis E virus, Viral factory, viral factories, medicine, Electron microscopy, Humans, AlphaFold2, Molecular Biology, 030304 developmental biology, Pharmacology, 0303 health sciences, 030306 microbiology, ORF2 capsid protein, Antibodies, Monoclonal, virus diseases, Biological Transport, Cell Biology, medicine.disease, Virology, digestive system diseases, 3. Good health, Endocytic recycling compartment, Rab11, Capsid, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Molecular Medicine, Capsid Proteins, Viral hepatitis, Viral Replication Compartments
Abstract: Background & AimsAlthough Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms.MethodsWe generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC).ResultsOne of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC.ConclusionsOur study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.Lay summaryHepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide but many steps of its lifecycle are still elusive. Thanks to the development of new antibodies that recognize the different forms of the HEV capsid protein, we were able to visualize vesicular and tubular structures that were established by the virus in the host cell. In addition, extensive efforts to identify these structures led us to conclude that HEV hijacks the endocytic recycling compartment of the cell to form this network of vesicles and tubules, which might be the hallmark of HEV infection.
Published: 2022

2. Broadly neutralizing anti-HIV-1 antibodies tether viral particles at the surface of infected cells

Author: Jérémy Dufloo, Cyril Planchais, Stéphane Frémont, Valérie Lorin, Florence Guivel-Benhassine, Karl Stefic, Nicoletta Casartelli, Arnaud Echard, Philippe Roingeard, Hugo Mouquet, Olivier Schwartz, Timothée Bruel, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), École Doctorale Bio Sorbonne Paris Cité [Paris] (ED562 - BioSPC), Université Sorbonne Paris Cité (USPC)-Université Paris Cité (UPCité), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), O.S. is funded by Institut Pasteur, Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS), Sidaction, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), 'TIMTAMDEN' ANR-14-CE14-0029, 'CHIKV-Viro-Immuno' ANR-14-CE14-0015-01, and the Gilead HIV cure program. J.D. is funded by a Ph.D grant from the French Ministry of Higher Education and Research. H.M. is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69-01), INSERM, ANRS, and Gilead HIV cure program. C.P. was supported by an ANRS fellowship. A.E. and S.F. are funded by Institut Pasteur, CNRS, and ANRS (ANRS-21020 AP2020-2). P.R. is funded by INSERM, Université de Tours and ANRS., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Vaccine Research Institute (VRI), Institut Pasteur, Agence Nationale de la Recherche (France), Ministére de l'Education Nationale de la Recherche et de la Technologie (France), and Université de Tours
Subjects: MESH: Epitopes, T-Lymphocytes, Science, viruses, General Physics and Astronomy, HIV Infections, MESH: Host Microbial Interactions, HIV Antibodies, General Biochemistry, Genetics and Molecular Biology, Cell Line, MESH: Antibodies, Neutralizing, MESH: HIV-1, Epitopes, MESH: Antibody-Dependent Cell Cytotoxicity, Humans, MESH: Broadly Neutralizing Antibodies, Multidisciplinary, MESH: Humans, Host Microbial Interactions, MESH: HIV Antibodies, Antibody-Dependent Cell Cytotoxicity, Virion, env Gene Products, Human Immunodeficiency Virus, virus diseases, General Chemistry, MESH: HIV Infections, Antibodies, Neutralizing, MESH: Cell Line, MESH: T-Lymphocytes, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HIV-1, MESH: Virion, MESH: env Gene Products, Human Immunodeficiency Virus, Broadly Neutralizing Antibodies
Abstract: Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) are promising molecules for therapeutic or prophylactic interventions. Beyond neutralization, bNAbs exert Fc-dependent functions including antibody-dependent cellular cytotoxicity and activation of the complement. Here, we show that a subset of bNAbs targeting the CD4 binding site and the V1/V2 or V3 loops inhibit viral release from infected cells. We combined immunofluorescence, scanning electron microscopy, transmission electron microscopy and immunogold staining to reveal that some bNAbs form large aggregates of virions at the surface of infected cells. This activity correlates with the capacity of bNAbs to bind to Env at the cell surface and to neutralize cell-free viral particles. We further show that antibody bivalency is required for viral retention, and that aggregated virions are neutralized. We have thus identified an additional antiviral activity of bNAbs, which block HIV-1 release by tethering viral particles at the surface of infected cells., O.S. is funded by Institut Pasteur, Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS), Sidaction, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), “TIMTAMDEN” ANR-14-CE14-0029, “CHIKV-Viro-Immuno” ANR-14-CE14-0015-01, and the Gilead HIV cure program. J.D. is funded by a Ph.D grant from the French Ministry of Higher Education and Research. H.M. is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69-01), INSERM, ANRS, and Gilead HIV cure program. C.P. was supported by an ANRS fellowship. A.E. and S.F. are funded by Institut Pasteur, CNRS, and ANRS (ANRS-21020 AP2020-2). P.R. is funded by INSERM, Université de Tours and ANRS.
Published: 2022

3. Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe

Author: Vincent Aucagne, Mylène Wartenberg, Gilles Lalmanach, Jean-Baptiste Madinier, Seda Seren, Fabien Lecaille, Guy Ilango, Ahlame Saidi, Brice Korkmaz, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Aucagne, Vincent, and Lalmanach, Gilles
Subjects: Proteinase 3, Spectrophotometry, Infrared, Cell Survival, Neutrophils, Myeloblastin, Biomedical Engineering, Pharmaceutical Science, Bioengineering, Near-infrared fluorescence (NIRF), Cathepsin G, Gene Expression Regulation, Enzymologic, Neutrophil Activation, Cathepsin, law.invention, chemistry.chemical_compound, Confocal microscopy, law, Elastase, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Dark quencher, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Fluorescent Dyes, Pharmacology, Microscopy, Confocal, Molecular Structure, biology, Neutrophil serine protease (NSP), Ionomycin, Organic Chemistry, Fluorescence, Förster resonance energy transfer, chemistry, Neutrophil elastase, biology.protein, Biophysics, Norvaline, Biotechnology
Abstract: International audience; A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440 000 ± 5500 M-1·s-1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.
Published: 2021

4. Structural modifications of the chorioallantoic membrane and the inner eggshell during chicken embryonic development

Author: Halgrain, Maëva, Bernardet, Nelly, Georgeault, Sonia, Hincke, Maxwell, Réhault-Godbert, Sophie, REHAULT-GODBERT, Sophie, Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), University of Ottawa [Ottawa], LE STUDIUM (LE STUDIUM), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de recherche pour le développement [IRD] : UR-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio]
Abstract: International audience; In oviparous species, the egg contains protective systems and provides energy and nutrients that support the development of the embryo until hatching. During embryonic development, extraembryonic structures are vital to assimilate egg nutrients, to protect the embryo, to ensure gaseous exchange and to accumulate metabolic waste. The chorioallantoic membrane (CAM), which develops in close contact with the inner eggshell from day 5 of incubation, is a highly vascularized structure. The CAM is located between the eggshell membranes (ESM, outer side) and the allantoic fluid that collects embryonic waste (inner side). The CAM was reported to be involved in decalcification of the inner eggshell and mineral transport from the eggshell to the embryo (for mineralization of its skeleton), in acid-base homeostasis, and in ion and H2O reabsorption from the allantoic fluid. To decipher the kinetics of its structural maturation, we compared the structure of the inner and outer surfaces of the CAM by electron microscopy. In parallel, ESM and the eggshell were analyzed to better appreciate the impact of CAM development on its surrounding structures. Eggs from Ross 308 broiler hens were incubated for 11 or 15 days (ED11 vs ED15, early and mature stage of CAM development, respectively). At each time point, eggs were broken, and the eggshell, ESM and the CAM were gently separated for analysis by scanning electron microscopy. Images corresponding to the cross-section and the inner side of the eggshell, eggshell membranes (ESM, outer and inner sides) and CAM (outer and inner sides) were obtained. At ED15, results showed that the inner side of the eggshell is degraded (decalcification) during embryonic development while the mammillary knobs of the eggshell remain tightly attached to the external membrane of the ESM. The CAM also undergoes major structural changes with some villous cells that emerge from its outer surface to reach the eggshell membrane and the eggshell. The molecular components associated with eggshell decalcification and mineral transport from the eggshell to the embryo via CAM’s vasculature remain essentially unknown. This study has been recently complemented by a transcriptomic analysis of the CAM (RNA sequencing) at these two developmental stages. Altogether, we believe that the combined findings of this work will lead to better understanding of CAM functions that are vital for embryonic development and skeletal mineralization during incubation.
Published: 2022

5. Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate

Author: Sylvie M. Roche, Julien Burlaud-Gaillard, Isabelle Virlogeux-Payant, Sébastien Holbert, Agnès Wiedemann, Stéphane Méresse, Philippe Velge, Sonia Georgeault, Emilie Barilleau, Jérôme Trotereau, Infectiologie et Santé Publique (UMR ISP), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), INRAE, Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and HOLBERT, Sébastien
Subjects: Salmonella, Cell signaling, Cell biology, Science, [SDV]Life Sciences [q-bio], Vacuole, medicine.disease_cause, Microbiology, Article, Type three secretion system, T3SS1, invasion factors, 03 medical and health sciences, Mice, Bacterial Proteins, medicine, Type III Secretion Systems, Animals, Cytoskeleton, 030304 developmental biology, 0303 health sciences, Multidisciplinary, 030306 microbiology, Effector, Chemistry, Pathogenicity island, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV] Life Sciences [q-bio], Salmonella Infections, Vacuoles, Salmonella Typhimurium, Hepatocytes, Medicine, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Intracellular
Abstract: Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.
Published: 2021

6. Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress

Author: Sébastien Eymieux, Rustem Uzbekov, Yves Rouillé, Emmanuelle Blanchard, Christophe Hourioux, Jean Dubuisson, Sandrine Belouzard, Philippe Roingeard, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)
Subjects: QH301-705.5, SARS-CoV-2, Secretory Vesicles, viruses, [SDV]Life Sciences [q-bio], Virion, coronavirus, virus egress, virus diseases, COVID-19, Virus Replication, Article, SARS-CoV-2 coronavirus COVID-19 virus egress virus release transmission electron microscopy serial sections, Microscopy, Electron, Transmission, Chlorocebus aethiops, virus release, transmission electron microscopy, Animals, serial sections, Biology (General), Vero Cells
Abstract: International audience; The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.
Published: 2021

7. Les usages de Twitter au festival de Cannes. Pratiques numériques et construction des opinions esthétiques

Author: Dougados, Myriam, Fabiani, Jean-Louis, Gaillard, Julien, Portilla, Yonathan, Avignon Université (AU), École des hautes études en sciences sociales (EHESS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Laboratoire Informatique d'Avignon (LIA), Centre d'Enseignement et de Recherche en Informatique - CERI-Avignon Université (AU), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Centre Norbert Elias (CNELIAS), École des hautes études en sciences sociales (EHESS)-Avignon Université (AU)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Avignon Université (AU)-Centre d'Enseignement et de Recherche en Informatique - CERI
Subjects: social networks, lexicology, [SHS.INFO]Humanities and Social Sciences/Library and information sciences, Twitter, public, gusto, goût, lexicología, [SHS.MUSEO]Humanities and Social Sciences/Cultural heritage and museology, festival, taste, lexicologie, públicos, audience, réseaux sociaux, redes sociales
Abstract: International audience; This article is a contribution to the analysis of the effects of social networks on the shaping of taste and opinions concerning cultural matters. Taking as an example the Twitter usage of thirty-two professional partners of Vodkaster who play with each other on the Cannes stage and using mainly lexical analysis, one can shed light on three structural concepts: the digital outsider – his deviance being a central feature of the event, leadership – based on faithful readership, and the fourth circle of spectators who experience the festival by proxy. Thus, digital social networks are both an easily mobilized resource for the analysis of audience and a specific form of festival-goer’s sociability.; Este artículo es una contribución al análisis de los efectos de las redes sociales sobre la formación de los gustos y de las opiniones en materia cultural. Tomando el ejemplo de los usos de Twitter en el festival de Cannes de treinta y dos profesionales (socios de Vodkaster) que tocan juntos en la escena local, y apoyados fundamentalmente en un análisis lexical, podemos dar a luz tres nociones estructurantes : la de outsider numérico (la desviación constitutiva del acontecimiento), la de liderazgo fundada sobre la fidelidad de los lectores y la del cuarto círculo de espectadores que construye su experiencia del festival en su nombre. Las redes sociales constituyen a la vez un recurso fácilmente explotable para el análisis de los públicos y una forma específica de la sociabilidad del festival.; Cet article est une contribution à l’analyse des effets des réseaux sociaux sur la formation des goûts et des opinions en matière culturelle. En prenant l’exemple des usages de Twitter au Festival de Cannes par trente-deux professionnels partenaires de Vodkaster qui jouent ensemble sur la scène cannoise et en s’appuyant principalement sur une analyse lexicale, on peut mettre au jour trois notions structurantes : celle d’outsider numérique – la déviance étant constitutive de l’événement –, celle de leadership fondée sur la fidélité du lectorat, et celle d’un quatrième cercle de spectateurs qui construit son expérience du Festival par procuration. Les réseaux sociaux numériques constituent ainsi à la fois une ressource aisément exploitable pour l’analyse des publics et une forme spécifique de la sociabilité festivalière.
Published: 2021

8. 12/111phiA Prophage Domestication Is Associated with Autoaggregation and Increased Ability to Produce Biofilm in Streptococcus agalactiae

Author: Julien Gaillard, Laurent Mereghetti, Seydina M. Diene, Roland Quentin, Nathalie van der Mee-Marquet, Patrice Francois, Adélaïde Renard, Houssein Gbaguidi-Haore, Luka Courtier-Martinez, Infectiologie et Santé Publique (UMR ISP), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Microbes évolution phylogénie et infections (MEPHI), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Service d’Hygiène Hospitalière, Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Laboratoire de Recherche Génomique, Geneva University Hospital (HUG)-Centre médical universitaire de Genève (CMU), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU), Laboratoire Chrono-environnement - CNRS - UBFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Centre Hospitalier Régional Universitaire [Besançon] (CHRU Besançon), Service de bactériologie et d'hygiène hospitalière [Tours], Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université, Genomic research lab, Geneva University Hospital (HUG), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), and Laboratoire Chrono-environnement - CNRS - UFC (UMR 6249) (LCE)
Subjects: Microbiology (medical), QH301-705.5, Streptococcus agalactiae, Biology, medicine.disease_cause, Cell morphology, Microbiology, Article, biofilm, 03 medical and health sciences, Neonate, autoaggregation, Virology, Lysogenic cycle, medicine, phage, Pathogenicity, pathogenicity, Biology (General), Autoaggregation, ORFS, Prophage, 030304 developmental biology, ddc:616, 0303 health sciences, 030306 microbiology, Biofilm, Toxin-antitoxin system, 3. Good health, Bacterial adhesin, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Phage, neonate, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; CC17 Streptococcus agalactiae carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.
Published: 2021

9. Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium

Author: Barilleau, Emilie, Védrine, Mégane, Koczerka, Michael, Burlaud-Gaillard, Julien, Kempf, Florent, Grépinet, Olivier, Virlogeux-Payant, Isabelle, Velge, Philippe, Wiedemann, Agnès, Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut de Recherche en Santé Digestive (IRSD ), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-15-IFEC-0003,Sal host trop,Understanding the Human-Restricted Host Tropism of Typhoidal Salmonella(2015), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: Salmonella typhimurium, Zipper-like entry pathway, Research, Integrin beta1, Fibroblasts, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Microbiology, QR1-502, Cell Line, Mice, Invasion, Salmonella, Outer membrane protein, PagN, Salmonella Infections, Type III Secretion Systems, Animals, Humans, Actin, Bacterial Outer Membrane Proteins
Abstract: Background. Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear.Results. Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of β1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in β1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-β1 integrin antibody, supporting the hypothesis that β1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization.Conclusions. Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.
Published: 2021

10. Combination of β‐(1, 3)‐D‐glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia

Author: Adrien Lemaignen, Victor Mercier, Sébastien Eymieux, Adélaïde Chesnay, Alain Goudeau, E. Bailly, José Bras‐Cachinho, Guillaume Desoubeaux, Marie-Alix De Kyvon, Parastou Moshiri, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), CHU Trousseau [Tours], Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)
Subjects: Male, calcofluor-blue, 0301 basic medicine, medicine.medical_specialty, beta-Glucans, 030106 microbiology, β 1 3 d glucan, Dermatology, Pneumocystis carinii, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Gastroenterology, law.invention, 030207 dermatology & venereal diseases, 03 medical and health sciences, 0302 clinical medicine, Antigen, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Nasopharyngeal aspirate, law, Nasopharynx, Internal medicine, medicine, Humans, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, Polymerase chain reaction, Aged, business.industry, Pneumonia, Pneumocystis, fungal infection, Pneumocystis jirovecii Pneumonia, General Medicine, Middle Aged, medicine.disease, 3. Good health, Staining, respiratory samples, Pneumonia, Infectious Diseases, Real-time polymerase chain reaction, Female, Uvitex, business, Bronchoalveolar Lavage Fluid
Abstract: International audience; BACKGROUND: Currently, the biological diagnosis of Pneumocystis jirovecii pneumonia (PjP infection) usually relies on microbiological investigations in bronchial-alveolar lavage fluid (BALF) by conventional staining methods and/or molecular biology. However, bronchial-alveolar lavage is sometimes complicated to manage, especially in weakened patients. Therefore, alternative clinical samples-easier to collect-are warranted in such specific contexts.OBJECTIVE: Over a four-year period, diagnostic performance of an original method based on combination of quantitative real-time polymerase chain reaction (qPCR) in nasopharyngeal aspirate (NPA) with measurement of β-(1, 3)-D-glucan antigen (BDG) in serum was prospectively assessed in a single centre.PATIENTS/METHODS: Results were compared with those obtained in BALF through direct staining methods and qPCR. True positives were defined by an independent committee based on clinical, radiological and biological data. Overall, 48 individuals with a definitive diagnosis of PjP infection were included, and 48 controls were selected upon matching for age, sex and underlying disease(s).RESULTS: qPCR results were strongly correlated between BALF and NPA (P < .0001). Altogether, greater diagnostic performance was achieved when establishing the positive cut-off of BDG antigen at 143 pg/mL. In such conditions, sensitivity of the testing based on either positive BDG measurement or positive qPCR in NPA was then calculated at 93.75%, 95% CI [82.37%-98.40%], and specificity at 97.87%, 95% CI [87.66%-100.00%].CONCLUSIONS: Further validation through multicentre studies is now required, especially for establishing clear cut-offs. However, one could already state that combination of qPCR in the NPA with BDG measurement in serum may be a valuable substitute for BALF examination.
Published: 2019

11. Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release

Author: Sébastien Eymieux, Jean Dubuisson, Yves Rouillé, Manuel Rosa-Calatrava, Sandrine Belouzard, Philippe Roingeard, Karin Séron, Emmanuelle Blanchard, Olivier Terrier, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours (UT), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Virology and human respiratory Pathologies - Virology and human respiratory Pathologies (VirPath), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-21-COVR-0022,ANTICOV,Valeur pronostique des auto-anticorps ANTi-Interféron de type I chez les patients pris en charge pour un syndrome de détresse respiratoire aiguë associé au COVID-19 : une étude de cohorte observationnelle multicentriq(2021), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Morphogénèse et antigénicité du VIH et du virus des Hépatites [MAVIVH - U1259 Inserm - CHRU Tours ], Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], and Dubuisson, Jean
Subjects: Virus Replication, Exocytosis, Cell Line, 03 medical and health sciences, Cellular and Molecular Neuroscience, Microscopy, Electron, Transmission, Viral factory, virus/cell interactions, Chlorocebus aethiops, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Electron microscopy, Animals, Nuclear pore, Vero Cells, Molecular Biology, Virus Release, 030304 developmental biology, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Pharmacology, 0303 health sciences, Chemistry, SARS-CoV-2, Vesicle, 030302 biochemistry & molecular biology, Cell Biology, Virus/cell interactions, Cell biology, Covid-19, Viral replication, Spike Glycoprotein, Coronavirus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Ultrastructure, Vero cell, Molecular Medicine, Original Article, Viral Replication Compartments
Abstract: International audience; Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.
Published: 2021

12. Non-permissive human conventional CD1c+ dendritic cells enable trans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization

Author: Steven Nedellec, Julien Burlaud-Gaillard, Philippe Hulin, Dorian McIlroy, Alexandra Garcia, Cécile Braudeau, Mathieu Sikorski, Antoine Touzé, Christine Kandel-Aznar, Pauline Gaboriaud, Flora Coulon, Matthieu Giraud, Régis Josien, Joëlle Veziers, Julien Branchereau, Franck Halary, Karine Renaudin, Cécile Peltier, Céline Bressollette-Bodin, Chanteloup, Nathalie Katy, Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), UMR 1064/facility TRIP/UMRS3556, Center for Research in Transplantation and Immunology, Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d’Immunologie [CHU Nantes] (Centre d’Immunomonitorage Nantes Atlantique - CIMNA), Centre hospitalier universitaire de Nantes (CHU Nantes), Service d’Anatomie et Cytologie Pathologiques [CHU Nantes], Cellular and Tissular Imaging Core Facility of Nantes (MicroPICell), Université de Nantes (UN), Structure fédérative de recherche François Bonamy (SFR François Bonamy), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), Service d'Urologie [Nantes], Service de Transplantations rénales, Regenerative Medicine and Skeleton (RMeS), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), PHU 4 OTONN [CHU Nantes], Université de Nantes - UFR Odontologie, Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Service de virologie [CHU Nantes], Fondation d'Entreprises Progreffe, ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), Regenerative Medicine and Skeleton research lab (RMeS), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: Physiology, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Endocytic cycle, Kidney, Virus Replication, Monocytes, Virions, Antigens, CD1, Spectrum Analysis Techniques, Animal Cells, Medicine and Health Sciences, Biology (General), 0303 health sciences, Secretory Pathway, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Hematopoietic stem cell, Immunosuppression, Flow Cytometry, Endocytosis, Body Fluids, 3. Good health, [SDV] Life Sciences [q-bio], Blood, medicine.anatomical_structure, Cell Processes, Spectrophotometry, Cytophotometry, Cellular Types, Anatomy, Cellular Structures and Organelles, Research Article, QH301-705.5, Immune Cells, Immunology, Antigen-Presenting Cells, Viral Structure, Biology, Research and Analysis Methods, Microbiology, Nephropathy, 03 medical and health sciences, Virology, Parenchyma, Genetics, medicine, Humans, Vesicles, Molecular Biology Techniques, Molecular Biology, Glycoproteins, 030304 developmental biology, Polyomavirus Infections, 030306 microbiology, Biology and Life Sciences, Kidneys, Epithelial Cells, Cell Biology, Dendritic Cells, Renal System, RC581-607, medicine.disease, Antibodies, Neutralizing, Epithelium, Tumor Virus Infections, BK Virus, Parasitology, Immunologic diseases. Allergy, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Cloning
Abstract: The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+ mDC involvement in the BKPyV infection as a promoter of viral spreading., Author summary Dr Sylvia Gardner first discovered the BK polyomavirus (BKPyV) in the urine of a kidney-transplant recipient in 1970. In the 1990’s, the widespread use of potent immunosuppressive drugs such as tacrolimus, sirolimus or mycophenolate mofetil led to the emergence of BKPyV nephropathy. Recently, various studies reported a specific influx of myeloid dendritic cells (mDCs) in the renal tissue of kidney-transplant patients who were diagnosed with a BKPyV nephropathy. MDCs are immune cells both residing in tissues and migrating to other organs or compartments like the blood when changes in their environment occur. Their main functions are the detection of danger signals such as pathogens or tumors and the processing of antigens to prime naïve specific effectors of the adaptive immune response. Although anti-BKPyV cellular immune responses have been investigated in post-transplant recipients as well as healthy individuals, supporting an active role of mDCs little is known about how mDCs and BKPyV interact with each other. Our study provides the basis to understand the role played by mDCs in virus capture through an unprecedented endocytic mechanism and possibly in viral protection from neutralization by specific antibodies. Moreover, we showed that mDCs are unable to sense BKPyV particles or BKPyV-infected dying cells as a danger signal, supporting the view that other DC subsets might act as the true antigen presenting cells that promote the adaptive immune response against BKPyV infection.
Published: 2021

13. Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

Author: Durand, Stéphanie, Seigneuret, Florian, Burlaud-Gaillard, Julien, Lemoine, Roxane, Tassi, Marc-Florent, Moreau, Alain, Mougel, Marylène, Roingeard, Philippe, Tauber, Clovis, de Rocquigny, Hugues, Herrada, Anthony, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), EA4245 - Transplantation, Immunologie, Inflammation [Tours] (T2i), Université de Tours (UT), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), and Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: SL, stem loop, ZF, zinc finger, assembly, PT, posttransfection, viruses, [SDV]Life Sciences [q-bio], Gene Products, gag, RSV, Rous Sarcoma Virus, Microscopy, Electron, Transmission, transmission electron microscopy, morphometric TEM analysis, eGFP, enhanced GFP, Gag, TEM, Transmission Electron Microscopy, Virus Assembly, Genomics, genomic RNA, [SDV] Life Sciences [q-bio], gRNA, genomic RNA, Nucleoproteins, PM, plasma membrane, HIV-1, RNA, Viral, BSA, bovine serum albumin, 5' Untranslated Regions, Research Article, RNA, Guide, Kinetoplastida
Abstract: International audience; In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.
Published: 2021

14. Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites

Author: Alexandre Bougdour, Fabrice Laurent, Christopher Swale, Tiffany Pezier, Valeria Bellini, Mohamed-Ali Hakimi, Sonia Georgeault, Marie-Pierre Brenier-Pinchart, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), ANR-19-CE15-0026,ToxoP53,Interférence de la protéine GRA16 de Toxoplasma avec la voie HAUSP/p53 : une manière de détourner la régulation médiée par p53 du métabolisme et de la signalisation immunitaire lors de la toxoplasmose ?(2019), ANR-18-CE15-0023,HostQuest,Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée(2018), European Project: 614880,Hosting Toxo, CCSD, Accord Elsevier, Interférence de la protéine GRA16 de Toxoplasma avec la voie HAUSP/p53 : une manière de détourner la régulation médiée par p53 du métabolisme et de la signalisation immunitaire lors de la toxoplasmose ? - - ToxoP532019 - ANR-19-CE15-0026 - AAPG2019 - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Stratégies de Persistence de Toxoplasma gondii : Conquérir la Cellule Hôte et Echapper à la Réponse Immune Innée - - HostQuest2018 - ANR-18-CE15-0023 - AAPG2018 - VALID, ERC Consolidator grant - Hosting Toxo - 614880 - INCOMING, Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: 0301 basic medicine, Polyadenylation, Protein subunit, [SDV]Life Sciences [q-bio], 02 engineering and technology, Computational biology, Microbiology Parasite, Microbiology, Article, Apicomplexa, 03 medical and health sciences, parasitic diseases, medicine, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, lcsh:Science, Molecular Biology, Multidisciplinary, biology, Toxoplasma gondii, Cryptosporidium, 021001 nanoscience & nanotechnology, biology.organism_classification, medicine.disease, Forward genetics, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Drug development, Molecular Medicine, lcsh:Q, 0210 nano-technology, Malaria, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract: Summary Boron-containing compounds represent a promising class of molecules with proven efficacy against a wide range of pathogens, including apicomplexan parasites. Following lead optimization, the benzoxaborole AN13762 was identified as a preclinical candidate against the human malaria parasite, yet the molecular target remained uncertain. Here, we uncovered the parasiticidal mechanisms of AN13762, by combining forward genetics with transcriptome sequencing and computational mutation discovery and using Toxoplasma gondii as a relevant model for Apicomplexa. AN13762 was shown to target TgCPSF3, the catalytic subunit of the pre-mRNA cleavage and polyadenylation complex, as the anti-pan-apicomplexan benzoxaborole compound, AN3661. However, unique mutations within the TgCPSF3 catalytic site conferring resistance to AN13762 do not confer cross-protection against AN3661, suggesting a divergent resistance mechanism. Finally, in agreement with the high sequence conservation of CPSF3 between Toxoplasma and Cryptosporidium, AN13762 shows oral efficacy in cryptosporidiosis mouse model, a disease for which new drug development is of high priority., Graphical Abstract, Highlights • AN13762 is active against T. gondii parasites • Parasites resistant to AN13762 harbor mutations within TgCPSF3 • Mutations within TgCPSF3 confer resistance to AN13762 • AN13762 offers an alternative for targeting CPSF3 in Toxoplasma and Cryptosporidium, Molecular Biology; Molecular Medicine; Microbiology; Microbiology Parasite
Published: 2020

15. Emergency department outcome of elderly patients assisted by professional home services, the EPIGER study

Author: Feral-Pierssens, Anne-Laure, Toury, Gustave, Sehimi, Fatima, Peschanski, Nicolas, Laribi, Saïd, Carpentier, Amélie, Kraif, Magali, Carbonnier, Clément, Duchateau, François-Xavier, Freund, Yonathan, Juvin, Philippe, EPIGER IRU-SFMU study group, Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Service des Urgences [CHU Saint-Antoine], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Hôpital Charles Nicolle [Rouen], Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Centre hospitalier [Valenciennes, Nord], Hôpital de la Timone [CHU - APHM] (TIMONE), Laboratoire interdisciplinaire d'évaluation des politiques publiques [Sciences Po] (LIEPP), Sciences Po (Sciences Po), Hôpital Raymond Poincaré [AP-HP], Service des Urgences [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), and Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)
Subjects: medicine.medical_specialty, Coping (psychology), [SDV]Life Sciences [q-bio], lcsh:Geriatrics, Lower risk, Home services, 03 medical and health sciences, 0302 clinical medicine, Elderly, Quality of life, Health care, medicine, Risk of mortality, Humans, Hospital Mortality, Prospective Studies, 030212 general & internal medicine, Prospective cohort study, Aged, Aged, 80 and over, business.industry, Mortality rate, 030208 emergency & critical care medicine, Emergency department, Healthcare access, 3. Good health, Hospitalization, lcsh:RC952-954.6, Loss of autonomy, Emergency medicine, Quality of Life, Emergency care, Geriatrics and Gerontology, Emergency Service, Hospital, business, Research Article
Abstract: Background For the elderly population living at home, the implementation of professional services tends to mitigate the effect of loss of autonomy and increases their quality of life. While helping in avoiding social isolation, home services could also be associated to different healthcare pathways. For elderly patients, Emergency Departments (EDs) are the main entrance to hospital where previous loss of autonomy is associated to worst hospital outcomes. Part of elderly patients visiting EDs are still admitted to hospital for having difficulties coping at home without presenting any acute medical issue. There is a lack of data concerning elderly patients visiting EDs assisted by home services. Our aim was to compare among elderly patients visiting ED those assisted by professional home services to those who do not in terms of emergency resources’ use and patients’ outcome. Methods A multicenter, prospective cohort study was performed in 124 French EDs during a 24-h period on March 2016.Consecutive patients living at home aged ≥80 years were included. The primary objective was to assess the risk of mortality for patients assisted by professional home services vs. those who were not. Secondary objectives included admission rate and specific admission rate for “having difficulties coping at home”. The primary endpoint was in-hospital mortality. Cox proportional-hazards regression model was used to test the association between professional home services and the primary endpoint. Multi variables logistic regressions were performed to assess secondary endpoints. Results One thousand one hundred sixty-eight patients were included, median age 86(83–89) years old,32% were assisted by professional home services. The overall in-hospital mortality rate was 7%. Assisted patients had more investigations performed. Home services were not associated with increased in-hospital mortality (HR = 1.34;95%CI [0.68–2.67]), nor with the admission rate (OR = 0.92;95%CI [0.65–1.30]). Assisted patients had a lower risk of being admitted for “having difficulties coping at home” (OR = 0.59;95%CI [0.38–0.92]). Conclusion Professional home services which assist one-third of elderly patients visiting EDs, were not associated to lower in-hospital mortality or to an increased admission rate. Assisted patients were associated to a lower risk of being admitted for «having difficulties coping at home».Professional home services could result in avoiding some admissions and their corollary complications. Trial registration Clinicaltrial.gov - NCT02900391, 09/14/2016, retrospectively registered
Published: 2020

16. A molecular determinant of West Nile virus secretion and morphology as a target for viral attenuation

Author: Justine, Basset, Julien, Burlaud-Gaillard, Maxence, Feher, Philippe, Roingeard, Félix A, Rey, Nathalie, Pardigon, Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP), Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats (CIBU), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Financial support was provided in part by Institut Carnot-Pasteur Microbes and Health, pardigon, nathalie, Institut Pasteur [Paris], Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Protein Conformation, alpha-Helical, viruses, Gene Expression, Antibodies, Viral, Mice, Viral Proteins, Cell Line, Tumor, Chlorocebus aethiops, Animals, membrane protein, Amino Acid Sequence, Vero Cells, attenuation, Neurons, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Mice, Inbred BALB C, Binding Sites, Membrane Glycoproteins, Sequence Homology, Amino Acid, Flavivirus, virus diseases, Antibodies, Neutralizing, Survival Analysis, Virus-Cell Interactions, secretion, Disease Models, Animal, Mutation, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Female, Protein Conformation, beta-Strand, Sequence Alignment, West Nile virus, West Nile Fever, Protein Binding
Abstract: West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health., West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains. IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.
Published: 2020

17. BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission

Author: Gilles Duverlie, Etienne Brochot, François Helle, Lynda Handala, Pierre-Ivan Raynal, Sandrine Castelain, Philippe Roingeard, Catherine François, Emmanuelle Blanchard, Virginie Morel, Véronique Descamps, Agents infectieux, résistance et chimiothérapie - UR UPJV 4294 (AGIR ), Université de Picardie Jules Verne (UPJV)-CHU Amiens-Picardie, Laboratoire de Virologie [CHU Amiens], CHU Amiens-Picardie, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), and DESSAIVRE, Louise
Subjects: en bloc transmission, MCPyV, [SDV]Life Sciences [q-bio], medicine.medical_treatment, Immunology, Cell, quasienveloped virus, polyomavirus, BKPyV, Biology, Endocytosis, Microbiology, Virus, Pathogenesis, 03 medical and health sciences, SV40, Extracellular Vesicles, Immune system, Virology, Chlorocebus aethiops, medicine, Animals, neutralizing antibodies, Spotlight, Vero Cells, 030304 developmental biology, 0303 health sciences, Polyomavirus Infections, 030306 microbiology, Immunosuppression, JCPyV, medicine.disease, Transplant rejection, Virus-Cell Interactions, [SDV] Life Sciences [q-bio], TSPyV, medicine.anatomical_structure, Insect Science, BK Virus, biology.protein, Antibody
Abstract: Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases., Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV. IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.
Published: 2020

18. Evidence That Zika Virus Is Transmitted by Breastfeeding to Newborn A129 (Ifnar1 Knock-Out) Mice and Is Able to Infect and Cross a Tight Monolayer of Human Intestinal Epithelial Cells

Author: Mathieu Hubert, Patricia Jeannin, Julien Burlaud-Gaillard, Philippe Roingeard, Antoine Gessain, Pierre-Emmanuel Ceccaldi, Aurore Vidy, Vidy-Roche, Aurore, Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Grand Programme Fédérateur LINMEC 'Links between Infection, Nutrition-Metabolism and Cancer,' Direction du Développement – SPPI, Institut Pasteur, Paris. Doctoral fellowship MH, French Ministry of Education and Research., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: Microbiology (medical), Aedes albopictus, breastfeeding, mouse model, [SDV]Life Sciences [q-bio], lcsh:QR1-502, Breast milk, Biology, Microbiology, lcsh:Microbiology, Zika virus, 03 medical and health sciences, Flaviviridae, oral transmission, intestinal epithelium, 030304 developmental biology, Original Research, entry site, 0303 health sciences, 030306 microbiology, Transmission (medicine), mother-to-child transmission, Outbreak, Transplacental, crossing, biology.organism_classification, Virology, 3. Good health, [SDV] Life Sciences [q-bio], Flavivirus
Abstract: International audience; Zika virus (ZIKV) belongs to the Flavivirus genus in the Flaviviridae family. Mainly transmitted via mosquito bites (Aedes aegypti, Aedes albopictus), ZIKV has been classified in the large category of arthropod-borne viruses, or arboviruses. However, during the past two outbreaks in French Polynesia (2013-2014) and Latin America (2015-2016), several cases of ZIKV human-to-human transmission were reported, either vertically via transplacental route but also horizontally after sexual intercourse. Interestingly, high viral burdens were detected in the colostrum and breast milk of infected women and mother-to-child transmission of ZIKV during breastfeeding was recently highlighted. In a previous study, we highlighted the implication of the mammary epithelium (blood-milk barrier) in ZIKV infectious particles excretion in breast milk. However, mechanisms of their further transmissibility to the newborn via oral route through contaminated breast milk remain unknown. In this study, we provide the first experimental proof-of-concept of the existence of the breastfeeding as a route for mother-to-child transmission of ZIKV and characterized the neonatal oral transmission in a well-established mouse model of ZIKV infection. From a mechanistical point-of-view, we demonstrated for the first time that ZIKV was able to infect and cross an in vitro model of tight human intestinal epithelium without altering its barrier integrity, permitting us to consider the gut as an entry site for ZIKV after oral exposure. By combining in vitro and in vivo experiments, this study strengthens the plausibility of mother-to-child transmission of ZIKV during breastfeeding and helps to better characterize underlying mechanisms, such as the crossing of the newborn intestinal epithelium by ZIKV. As a consequence, these data could serve as a basis for a reflection about the implementation of measures to prevent ZIKV transmission, while keeping in mind breastfeeding-associated benefits.
Published: 2020

19. Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid

Author: Hugues, de Rocquigny, Virgile, Rat, Florentin, Pastor, Jean Luc, Darlix, Christophe, Hourioux, Philippe, Roingeard, de Rocquigny, Hugues, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: assembly, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hepatitis B virus, HBc, phosphorylation, Virus Assembly, viruses, packaging, lcsh:QR1-502, Review, Arginine, Virus Replication, Hepatitis B Core Antigens, lcsh:Microbiology, chaperone activity, Nucleic Acids, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HBV, Humans, RNA
Abstract: International audience; The morphogenesis of Hepatitis B Virus (HBV) viral particles is nucleated by the oligomerization of HBc protein molecules, resulting in the formation of an icosahedral capsid shell containing the replication-competent nucleoprotein complex made of the viral polymerase and the pre-genomic RNA (pgRNA). HBc is a phospho-protein containing two distinct domains acting together throughout the viral replication cycle. The N-terminal domain, (residues 1-140), shown to self-assemble, is linked by a short flexible domain to the basic C-terminal domain (residues 150-183) that interacts with nucleic acids (NAs). In addition, the C-terminal domain contains a series of phospho-acceptor residues that undergo partial phosphorylation and de-phosphorylation during virus replication. This highly dynamic process governs the homeostatic charge that is essential for capsid stability, pgRNA packaging and to expose the C-terminal domain at the surface of the particles for cell trafficking. In this review, we discuss the roles of the N-terminal and C-terminal domains of HBc protein during HBV morphogenesis, focusing on how the C-terminal domain phosphorylation dynamics regulate its interaction with nucleic acids throughout the assembly and maturation of HBV particles.
Published: 2020

20. A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

Author: Deynoux, Margaux, Sunter, Nicola, Ducrocq, Elfi, Dakik, Hassan, Guibon, Roseline, Burlaud-Gaillard, Julien, Brisson, Lucie, Rouleux-Bonnin, Florence, le Nail, Louis-Romée, Hérault, Olivier, Domenech, Jorge, Roingeard, Philippe, Fromont, Gaëlle, Mazurier, Frédéric, ERL 7001 LNOx (Leukemic Niche & redOx metabolism / Niche leucémique et métabolisme redOx) (LNOx), Groupe innovation et ciblage cellulaire (GICC), EA 7501 [2018-...] (GICC EA 7501), Université de Tours (UT)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Anatomie et cytologie pathologique [Tours], Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Nutrition, croissance et cancer (U 1069) (N2C), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Chirurgie orthopédique [Tours], Service d'Hématologie biologique [Tours], The authors acknowledge the Ministry of Research (MD), the ARC Foundation (MD, HD), the French Society of Hematology (MD), the 'Ligue contre le Cancer (NS)', and the Lebanese south governate (HD) for their funding. This work was supported by the French Committees of the 'Ligue Contre le Cancer Grand-Ouest' [16 (Charente), 36 (Indre), 37 (Indre-et-Loire), 41 (Loire et Cher), and 86 (Vendée)] and the Région Centre Val de Loire (FM)., Bodescot, Myriam, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Centre National de la Recherche Scientifique (CNRS)-Microenvironnement des niches tumorales (CNRS GDR 3697 Micronit ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Groupe innovation et ciblage cellulaire (GICC), EA 7501 [2018-...] (GICC EA 7501), Université de Tours (UT)-Université de Tours (UT), Université de Tours-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, and Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)
Subjects: Autophagic Cell Death, Cell Lines, Science, Apoptosis, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Research and Analysis Methods, Cell Line, Mice, Animal Cells, Spheroids, Cellular, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Animals, Humans, Electron Microscopy, Hypoxia, Cell Aggregation, Cell Proliferation, Staining, Microscopy, Cell Death, Stem Cells, Biology and Life Sciences, Cell Staining, Mesenchymal Stem Cells, Cell Biology, Cell Dedifferentiation, Cell Hypoxia, Oxidative Stress, Cell Processes, Specimen Preparation and Treatment, embryonic structures, Medicine, Biological Cultures, Scanning Electron Microscopy, Cellular Types, Stem Cell Lines, Research Article
Abstract: International audience; Mesenchymal stem cells (MSC)-spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSC-spheroids began to shrink after 24 hours, the size of spheroids from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, like hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.
Published: 2020

21. Differentiation-dependent susceptibility of human muscle cells to Zika virus infection

Author: Gillian Butler-Browne, Jim Zoladek, Ingo Riederer, Quentin Giai Gianetto, Philippe Roingeard, Patricia Jeannin, Julien Burlaud-Gaillard, Pierre-Emmanuel Ceccaldi, Mariette Matondo, Valérie Choumet, Thibault Chaze, Vincent Legros, Vincent Mouly, Philippe V. Afonso, Antoine Gessain, Mariela-Natacha Gonzàlez, Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris], This work was supported by a Pasteur-Fiocruz grant. VL was supported by a fellowship from the 'Fondation pour la Recherche Médicale' (FRM), IR and MG by CNPq/Ciências sem Fronteiras program, CNPq/Brazilian National Institute of Science and Technology on Neuroimmunomodulation (INCT-NIM). FOCEM - Mercosur Structural Convergence Fund 03/11. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Pasteur [Paris] (IP), Matondo, Mariette, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), UMRS974, Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de Recherche en Myologie, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ)
Subjects: Proteomics, Pulmonology, RC955-962, Apoptosis, Pathology and Laboratory Medicine, Zika virus, 0302 clinical medicine, Medical Conditions, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Arctic medicine. Tropical medicine, MESH: Proteins, Chikungunya, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Zika Virus Infection, Stem Cells, MESH: Proteomics, 3. Good health, Medical Microbiology, Arboviral Infections, Viral Pathogens, Interferon Type I, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Public aspects of medicine, Cellular Types, MESH: Zika Virus, Microbiology, 03 medical and health sciences, Respiratory Disorders, MESH: Zika Virus Infection, Humans, Microbial Pathogens, MESH: Humans, Flaviviruses, MESH: Host-Pathogen Interactions, Public Health, Environmental and Occupational Health, Organisms, Biology and Life Sciences, Chikungunya Infection, Proteins, medicine.disease, Tropical Diseases, Virology, MESH: Cell Line, 030104 developmental biology, Biological Tissue, Interferon type I, Developmental Biology, 0301 basic medicine, RNA viruses, MESH: Cell Death, MESH: Interferon Type I, MESH: Muscle Cells, Viral Diseases, [SDV]Life Sciences [q-bio], Muscle Fibers, Skeletal, medicine.disease_cause, Virus Replication, Dengue fever, Myoblasts, Cytopathogenic Effect, Viral, Medicine and Health Sciences, Morphogenesis, Myocyte, Cytopathic effect, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Muscle Fibers, Skeletal, Cell Death, Myogenesis, Cell Differentiation, Muscle Differentiation, Infectious Diseases, Cell Processes, Viruses, Host-Pathogen Interactions, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Disease Susceptibility, Stem cell, RA1-1270, Pathogens, Anatomy, medicine.drug, Research Article, Neglected Tropical Diseases, MESH: Cell Differentiation, 030231 tropical medicine, Muscle Tissue, MESH: Disease Susceptibility, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Stem Cells, Biology, Cell Line, medicine, MESH: Myoblasts, Muscle Cells, MESH: Virus Replication, Cell Biology, Zika Virus, biology.organism_classification, MESH: Cytopathogenic Effect, Viral, Respiratory Infections
Abstract: Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis., Author summary Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, and may be involved in the disease manifestation. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human muscle stem cells (myoblasts) that can be differentiated into differentiated muscle cells (myotubes), we found that myoblasts can be infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection. Infection induced the death of infected cells. Protein levels 24h and 48h post-infection indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, muscle stem cells being susceptible while differentiated muscle cells are resistant. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences during ZIKV infection.
Published: 2020

22. Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

Author: Marc Tramier, Charline Herrscher, Pierre-Yvan Raynal, Stephanie Durand, Philippe Roingeard, Virgile Rat, Christophe Hourioux, Roxane Lemoine, Xavier Pinson, Florian Seigneuret, Julien Burlaud-Gaillard, Hugues de Rocquigny, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biosit : biologie, santé, innovation technologique (SFR UMS CNRS 3480 - INSERM 018), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Cellules Dendritiques, Immunomodulation et Greffes, Université de Tours (UT), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Institut National de la Santé et de la Recherche Médicale, Agence Nationale de Recherches sur le Sida et les Hépatites Virales, Tours University, Jonchère, Laurent, Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)
Subjects: DNA Replication, assembly, Hepatitis B virus, Viral capsid assembly, viruses, [SDV]Life Sciences [q-bio], Protein domain, Context (language use), Fluorescence correlation spectroscopy, Virus Replication, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Protein Domains, Structural Biology, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, capsid, HBV, Humans, FRET-FLIM, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, 0303 health sciences, Chemistry, HBc, Viral Core Proteins, Virus Assembly, DNA replication, virus diseases, Transfection, Hepatitis B, digestive system diseases, 3. Good health, [SDV] Life Sciences [q-bio], Capsid, Biophysics, RNA, Viral, 030217 neurology & neurosurgery
Abstract: International audience; Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.
Published: 2020

23. A previously unreported frameshift <scp>ATP</scp> 2C1 mutation in a generalized Hailey–Hailey disease

Author: Sarah Ventéjou, Thibault Kervarrec, Sophie Leducq, S. Duchatelet, Alain Hovnanian, Emmanuelle Blanchard, S. Eymieux, Laurent Machet, A. de Muret, J. Zaragoza, Service de dermatologie (CHRU de Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Service de Dermatologie [Orléans], Centre Hospitalier Régional d'Orléans (CHRO), Service de Pathologie [CHRU Tours], Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: Genetics, 0303 health sciences, business.industry, Dermatology, medicine.disease, Frameshift mutation, 030207 dermatology & venereal diseases, 03 medical and health sciences, Pemphigus, 0302 clinical medicine, Infectious Diseases, Hailey–Hailey disease, Familial Benign Pemphigus, Mutation (genetic algorithm), Medicine, business, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP.DERM]Life Sciences [q-bio]/Human health and pathology/Dermatology, 030304 developmental biology
Abstract: International audience
Published: 2019

24. The Atlastin ER-shaping proteins facilitate Zika virus replication

Author: Monel, Blandine, Rajah, Maaran Michael, Hafirassou, Mohamed-Lamine, Sid-Ahmed, Samy, Burlaud-Gaillard, Julien, Zhu, Peng-Peng, Nevers, Quentin, Buchrieser, Julian, Porrot, Françoise, Meunier, Cécile, Amraoui, Sonia, Chazal, Maxime, Salles, Audrey, Jouvenet, Nolwenn, Roingeard, Philippe, Blackstone, Craig, Amara, Ali, schwartz, olivier, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Ecole doctorale Bio Sorbonne Paris Cité (BioSPC) (ED 157), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5), Génomes, biologie cellulaire et thérapeutiques (GenCellDi (UMR_S_944)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Collège de France (CdF (institution))-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Cell Biology Section, Neurogenetics Branch [Bethesda], National Institute of Neurological Disorders and Stroke [Bethesda] (NINDS), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH), Génomique virale et vaccination, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège de France (CdF (institution))-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Virus et Immunité, Biologie cellulaire des infections virales, Collège de France (CdF)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Tours-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Technologie et Service BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), and Institut Pasteur [Paris]-Institut Pasteur [Paris]
Subjects: [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Abstract: International audience; The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by “implosive” cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.
Published: 2019

25. Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe

Author: Mylène Wartenberg, Ahlame Saidi, Mathieu Galibert, Vincent Aucagne, Alix Joulin-Giet, Gilles Lalmanach, Christopher J. Scott, Julien Burlaud-Gaillard, Agnès F. Delmas, Fabien Lecaille, Pathologies Respiratoires : Protéolyse et Aérosolthérapie, Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Queen's University [Belfast] (QUB), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Lalmanach, Gilles, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), and Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)
Subjects: 0301 basic medicine, THP-1 Cells, Macrophage, [SDV]Life Sciences [q-bio], Peptide, Near-infrared fluorescence (NIRF), 7. Clean energy, Biochemistry, Substrate Specificity, Cysteine cathepsin, Mice, 03 medical and health sciences, Fluorescence Resonance Energy Transfer, Extracellular, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Enzyme kinetics, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Fluorescent Dyes, Cathepsin S, chemistry.chemical_classification, Spectroscopy, Near-Infrared, CATS, 030102 biochemistry & molecular biology, biology, Chemistry, Macrophages, General Medicine, Förster resonance energy transfer (FRET), Probe, Cathepsins, Fluorescence, Molecular Imaging, Protease, 030104 developmental biology, Förster resonance energy transfer, Neutrophil elastase, biology.protein, Biophysics, Oligopeptides, Spleen
Abstract: International audience; We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2–8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M−1 s−1) and sensitive to low concentration of CatS (
Published: 2019

26. Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1

Author: Lucie Etienne, Julien Burlaud-Gaillard, George Savidis, Andrea Cimarelli, Li Zhong, Philippe Roingeard, Mathilde Delpeuch, Romain Appourchaux, Kevin Tartour, Abraham L. Brass, Interaction hôte pathogène lors de l'infection lentivirale – Host-Pathogen Interaction during Lentiviral Infection (LP2L), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Department of Microbiology and Physiological Systems, University of Massachusetts Medical School [Worcester] (UMASS), University of Massachusetts System (UMASS)-University of Massachusetts System (UMASS), Virus, pseudo-virus: Morphogénèse et Antigénicité, Université de Tours (UT)-EA3856, Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, and Université de Tours-EA3856
Subjects: Endosome, Immunology, Mutant, Cellular Response to Infection, Context (language use), Endosomes, Biology, Microbiology, Virus, 03 medical and health sciences, Protein Domains, Interferon, Virology, medicine, Humans, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Infectivity, 0303 health sciences, Protein Stability, Tetraspanin 30, Virus Assembly, 030302 biochemistry & molecular biology, Virion, Membrane Proteins, RNA-Binding Proteins, Transmembrane protein, 3. Good health, Cell biology, Cholesterol, HEK293 Cells, Insect Science, Mutation, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HIV-1, Intracellular, HeLa Cells, medicine.drug
Abstract: The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.
Published: 2019

27. Ultrasonic identification technique in recycling of lithium ion batteries

Author: Satyajit Phadke, Michiel Postema, Cuthbert Nyamupangedengu, Anthony Novell, Ayache Bouakaz, Rustem Uzbekov, Mérièm Anouti, Postema, Michiel, University of the Witwatersrand [Johannesburg] (WITS), LE STUDIUM (LE STUDIUM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de recherche pour le développement [IRD] : UR-Centre National de la Recherche Scientifique (CNRS), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physico-chimie des Matériaux et des Electrolytes pour l'Energie (PCM2E), Université de Tours (UT), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Lomonosov Moscow State University (MSU), Bureau de Recherches Géologiques et Minières (BRGM) (BRGM)-Centre national du machinisme agricole, du génie rural, des eaux et forêts (CEMAGREF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de recherche pour le développement [IRD] : UR-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Le Studium Loire Valley Institute for Advanced Studies, 45000 Orléans, France, Université de Tours, Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Département des Microscopies, plate-forme R.I.O. de microscopie électronique, Université Francois Rabelais [Tours], This project has been supported by LE STUDIUM Loire Valley Institute for Advanced Studies, Orléans & Tours, France with funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 665790, Bureau de Recherches Géologiques et Minières (BRGM) (BRGM)-Centre national du machinisme agricole, du génie rural, des eaux et forêts (CEMAGREF)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de recherche pour le développement [IRD] : UR-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: FOS: Computer and information sciences, Sound (cs.SD), Materials science, cathode separation, chemistry.chemical_element, FOS: Physical sciences, 02 engineering and technology, Applied Physics (physics.app-ph), 01 natural sciences, 7. Clean energy, Signal, Computer Science - Sound, Lithium-ion battery, law.invention, Ion, ultrasonic particle manipulation, law, Audio and Speech Processing (eess.AS), 0103 physical sciences, FOS: Electrical engineering, electronic engineering, information engineering, lithium ion battery recycling, cathode material identification, 010302 applied physics, [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], [SPI.ACOU] Engineering Sciences [physics]/Acoustics [physics.class-ph], Scattering, harmonic imaging, Physics - Applied Physics, 021001 nanoscience & nanotechnology, Cathode, [PHYS.MECA.ACOU]Physics [physics]/Mechanics [physics]/Acoustics [physics.class-ph], Chemical engineering, chemistry, Harmonic, [SDV.IB]Life Sciences [q-bio]/Bioengineering, Ultrasonic sensor, Lithium, 0210 nano-technology, Electrical Engineering and Systems Science - Audio and Speech Processing
Abstract: The recycling of lithium ion batteries has been mentioned as one of the near-future waste management necessities. In order for recycling to be economically viable, straightforward and cost effective techniques need to be developed to separate the individual materials in a composite electrode. Ultrasonic separation might be such a technique, provided that lithium ion battery microparticles respond predictably to a sound field. Lithium ion battery cathodes contain hydrophobic carbon. Owing to the incompressibility of a solid, the thin gaseous layer surrounding these hydrophobic particles must oscillate asymmetrically, when subjected to ultrasound. Consequently, the harmonic content of the ultrasound signal radiated from hydrophobic microparticles must be higher than that from hydrophilic microparticles with the same size. The question of whether the harmonic signal response generated by physical hydrophobic microparticles present in lithium ion battery cathodes is higher than the harmonic response of other component materials in the cathode is the focus of this paper. The scattering response of cathode materials subjected to 1-MHz ultrasound was measured and compared. The cathode materials C65, PVDF, and NMC respond differently to 1-MHz ultrasound. The superharmonic response of C65 has been attributed to asymmetric oscillations owing to its hydrophobicity. In addition, C65 hydrophobic microparticles might be suitable candidates for harmonic imaging., Comment: 4 pages, 6 figures, submitted as a Proceeding to the IEEE AFRICON 2019 conference
Published: 2019

28. Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety

Author: Sébastien Eymieux, Pierre-Ivan Raynal, Emmanuelle Blanchard, Philippe Roingeard, Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours (UT), Roingeard, Philippe, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours
Subjects: 0301 basic medicine, 030106 microbiology, viral diagnosis, Reviews, Context (language use), Review, virus, Biology, Virus, 03 medical and health sciences, Biosafety, Microscopy, Electron, Transmission, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Virology, Disease Transmission, Infectious, Animals, Humans, Technology, Pharmaceutical, ComputingMilieux_MISCELLANEOUS, viral safety, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, electron microscopy, Diagnostic Tests, Routine, Virion, Containment of Biohazards, 3. Good health, Virus detection, Highly sensitive, 030104 developmental biology, Infectious Diseases, Virus Diseases, Viruses, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Imaging technique, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Infectious agent
Abstract: Summary Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer‐scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme‐linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its “catch‐all” nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little‐known, difficult‐to‐detect viruses. In this context, the “catch‐all” properties of TEM make it possible to document the presence of viruses or virus‐like particles in these products.
Published: 2019

29. Oscillating endoskeletal antibubbles

Author: Nobuki Kudo, Rustem Uzbekov, Ryunosuke Matsumoto, Ri-Ichiro Shimizu, Craig Carlson, Nicole Anderton, Aurélie Deroubaix, Clement Penny, Poortinga, Albert T., Rubin, David M., Ayache Bouakaz, Michiel Postema, Hokkaido University [Sapporo, Japan], Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Lomonosov Moscow State University (MSU), University of the Witwatersrand [Johannesburg] (WITS), Eindhoven University of Technology [Eindhoven] (TU/e), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Postema, Michiel
Subjects: Soft Condensed Matter (cond-mat.soft), FOS: Physical sciences, Medical Physics (physics.med-ph), Condensed Matter - Soft Condensed Matter, [PHYS.MECA.ACOU] Physics [physics]/Mechanics [physics]/Acoustics [physics.class-ph], Physics - Medical Physics, ComputingMilieux_MISCELLANEOUS, [PHYS.MECA.ACOU]Physics [physics]/Mechanics [physics]/Acoustics [physics.class-ph]
Abstract: An antibubble is a gas bubble with a liquid droplet inside. Antibubbles have a potential role in ultrasonic imaging, ultrasound-guided drug delivery, and hydrocarbon leakage detection. The liquid core droplets inside antibubbles can be stabilised with a process called Pickering stability. Other systems proposed include spoke crystals to suspend the droplet or endoskeletal structures. In this study, the oscillatory response of microscopic endoskeletal antibubbles to ultrasound has been investigated, primarily to determine whether the presence of an endoskeleton attenuates the oscillation amplitude of the antibubble. Three emulsions containing (anti)bubbles were prepared for evaluation. Each emulsion was pipetted into the observation chamber of a high-speed microscopic observation system. A high-speed camera, operating at 10 million frames per second, was couples to the microscope. During camera recording, the materials were subjected to ultrasound pulses from a laboratory-assembled single-element transducer. The presence of an endoskeleton with droplets hampers the oscillation of antibubbles as compared to identical bubbles without endoskeleton and droplet content., Comment: 6 papers, 4 figures, submitted to the 40th Symposium on UltraSonic Electronics (USE2019)
Published: 2019

30. Treatment of Pseudomonas aeruginosa Biofilm Present in Endotracheal Tubes by Poly- l -Lysine

Author: Clémence Henry, Antoine Guillon, Virginie Hervé, Sonia Georgeault, Mustapha Si-Tahar, Delphine Fouquenet, Eric Morello, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Gonzalez, Loïc, Centre Hospitalier Régional Universitaire de Tours (CHRU de Tours), Université de Tours (UT), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, and MORELLO, Eric
Subjects: 0301 basic medicine, medicine.drug_class, Swine, [SDV]Life Sciences [q-bio], 030106 microbiology, Antibiotics, Lysine, medicine.disease_cause, biofilm, Microbiology, endotracheal tube, 03 medical and health sciences, ventilator-associated pneumonia, medicine, Intubation, Intratracheal, Animals, Humans, Pharmacology (medical), Polylysine, Experimental Therapeutics, Pharmacology, poly-l-lysine, Chemistry, Pseudomonas aeruginosa, Biofilm, Ventilator-associated pneumonia, Pneumonia, Ventilator-Associated, Bacteria Present, medicine.disease, Respiration, Artificial, In vitro, 3. Good health, Anti-Bacterial Agents, antibiofilm agent, [SDV] Life Sciences [q-bio], Pneumonia, Infectious Diseases, Biofilms, Microscopy, Electron, Scanning, Equipment Contamination
Abstract: International audience; The endotracheal tube (ETT) is an essential interface between the patient and ventilator in mechanically ventilated patients. However, a microbial biofilm is formed gradually on this tube and is associated with the development of ventilator-associated pneumonia. The bacteria present in the biofilm are more resistant to antibiotics, and current medical practices do not make it possible to eliminate. Pseudomonas aeruginosa is one of the leading pathogens that cause biofilm infections and ventilator-associated pneumonia. Poly-l-lysine (pLK) is a cationic polypeptide possessing antibacterial properties and mucolytic activity by compacting DNA. Here, we explored the antibiofilm activity of pLK to treat P. aeruginosa biofilms on ETTs while taking into consideration the necessary constraints for clinical translation in our experimental designs. First, we showed that pLK eradicates a P. aeruginosa biofilm formed in vitro on 96-well microplates. We further demonstrated that pLK alters bacterial membrane integrity, as revealed by scanning electron microscopy, and eventually eradicates biofilm formed either by reference or clinical strains of P. aeruginosa biofilms generated in vitro on ETTs. Second, we collected the ETT from patients with P. aeruginosa ventilator-associated pneumonia. We observed that a single dose of pLK is able to immediately disrupt the biofilm structure and kills more than 90% of bacteria present in the biofilm. Additionally, we did not observe any lung tolerance issue when the pLK solution was instilled into the ETT of ventilated pigs, an animal model particularly relevant to mimic invasive mechanical ventilation in humans. In conclusion, pLK appears as an innovative antibiofilm molecule, which could be applied in the ETT of mechanically ventilated patients.
Published: 2018

31. The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

Author: Emmanuelle, Blanchard, Philippe, Roingeard, Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Roingeard, Philippe, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours (UT), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), and Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, hepatitis C virus, Hepatitis C virus, Biology, medicine.disease_cause, 03 medical and health sciences, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Lipid droplet, virus/cell interactions, medicine, hepatocyte, membranous web, lcsh:QH301-705.5, Host cell membrane, liver tissue, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Innate immune system, medicine.diagnostic_test, Brief Report, Endoplasmic reticulum, General Medicine, Virology, In vitro, 3. Good health, 030104 developmental biology, medicine.anatomical_structure, lcsh:Biology (General), Liver biopsy, Hepatocyte, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; Host cell membrane rearrangements induced by the hepatitis C virus (HCV) have been exclusively studied in vitro. These studies have shown that HCV induces double-membrane vesicles (DMVs), which probably serve to separate replication sites from the cytoplasmic sensors of the innate immune response. We report for the first time the observation of HCV-induced membrane rearrangements in liver biopsy specimens from patients chronically infected with HCV. Unlike observations performed in vitro, the membranous web detected in liver tissue seems essentially made of clusters of single-membrane vesicles derived from the endoplasmic reticulum and close to lipid droplets. This suggests that the DMVs could be a hallmark of laboratory-adapted HCV strains, possibly due to their ability to achieve a high level of replication. Alternatively, the concealment of viral RNA in DMVs may be part of innate immune response mechanisms particularly developed in hepatoma cell lines cultured in vitro. In any case, this constitutes the first report showing the differences in the membranous web established by HCV in vitro and in vivo.
Published: 2018
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32. Asymmetric oscillations of endoskeletal antibubbles

Author: David M. Rubin, Michiel Postema, Craig S. Carlson, Rustem Uzbekov, Albert T. Poortinga, Nicole Anderton, Aurélie Deroubaix, Clement Penny, Ryunosuke Matsumoto, Nobuki Kudo, Ayache Bouakaz, Ri-ichiro Shimizu, Hokkaido University [Sapporo, Japan], Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Lomonosov Moscow State University (MSU), University of the Witwatersrand [Johannesburg] (WITS), Eindhoven University of Technology [Eindhoven] (TU/e), Imagerie et cerveau (iBrain - Inserm U1253 - UNIV Tours ), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Postema, Michiel, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), and Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 010302 applied physics, Physics, Physics and Astronomy (miscellaneous), General Engineering, General Physics and Astronomy, 02 engineering and technology, Mechanics, 021001 nanoscience & nanotechnology, 01 natural sciences, Antibubble, [PHYS.MECA.ACOU]Physics [physics]/Mechanics [physics]/Acoustics [physics.class-ph], 0103 physical sciences, [PHYS.MECA.ACOU] Physics [physics]/Mechanics [physics]/Acoustics [physics.class-ph], 0210 nano-technology, Harmonic oscillator
Abstract: International audience; Antibubbles have been under investigation as potential vehicles in ultrasound-guided drug delivery. It is assumed that antibubbles can expand unhampered, but cannot contract beyond the size of their inner core. In this study, this hypothesis was tested on endoskeletal antibubbles and reference bubbles. These were subjected to 3-cycle pulses of 1-MHz ultrasound, whilst being recorded with a high-speed camera operating at 10 million frames per second. At low acoustic amplitudes (200 kPa), antibubbles and bubbles oscillated symmetrically. At high acoustic amplitudes (1.00 MPa), antibubbles and bubbles oscillated asymmetrically, but antibubbles significantly more so than bubbles. Furthermore, fragmentation and core release were observed at these amplitudes. This finding may have implications for ultrasound-guided drug delivery using antibubbles.
Published: 2020

33. The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

Author: Paris, Joris, Tobaly-Tapiero, Joëlle, Giron, Marie-Lou, Burlaud-Gaillard, Julien, Buseyne, Florence, Roingeard, Philippe, Lesage, Pascale, Zamborlini, Alessia, Saïb, Ali, Pathologie cellulaire : aspects moléculaires et viraux / Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), This study was supported by CNRS, INSERM, Université Paris Diderot Sorbonne Paris Cité, CNAM and ANR (ANR-12-BSV3-0016 to AS, ANR-15-CE15-0008 to AS and FB)., We thank Axel Rethwilm and Dirk Lindemann for FV reagents, Mark Bedford for GFP-PRMTs plasmids, Christelle Doliger, Sophie Duchez and Niclas Setterblad at the Imaging, Cell selection and Genomics Department of the Institut Universitaire d’Hématologie for confocal microscopy, Claudine Pique for critical reading of the manuscript., ANR-12-BSV3-0016,PTPs,Le rôle de l'exportation et de la maturation d'ARN messagers dans la production des produits pionniers de traduction (PTPs) dans la voie du CMH de la classe I.(2012), ANR-15-CE15-0008,REEMFOAMY,L'infection humaine par les virus foamy simiens zoonotiques : rôle des facteurs virologiques et immunologiques dans la restrcition de l'emergence virale(2015), Buseyne, Florence, BLANC - Le rôle de l'exportation et de la maturation d'ARN messagers dans la production des produits pionniers de traduction (PTPs) dans la voie du CMH de la classe I. - - PTPs2012 - ANR-12-BSV3-0016 - BLANC - VALID, L'infection humaine par les virus foamy simiens zoonotiques : rôle des facteurs virologiques et immunologiques dans la restrcition de l'emergence virale - - REEMFOAMY2015 - ANR-15-CE15-0008 - AAPG2015 - VALID, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]
Subjects: Protein-Arginine N-Methyltransferases, viruses, PRMT, MESH: Protein-Arginine N-Methyltransferases, Amino Acid Motifs, Nuclear Localization Signals, MESH: Nuclear Localization Signals, MESH: Amino Acid Sequence, MESH: Amino Acid Motifs, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Evolution, Molecular, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Gag, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, MESH: Arginine, Chromatin, Nuclear trafficking, Protein Transport, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, MESH: Repressor Proteins, MESH: Gene Products, gag, [SDV.IMM.IA] Life Sciences [q-bio]/Immunology/Adaptive immunology, MESH: Retroviridae Infections, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Foamy virus, Post-translational modification, Chromatin-binding, Protein Binding, lcsh:Immunologic diseases. Allergy, MESH: Cell Nucleus, MESH: Protein Transport, Gene Products, gag, Arginine, Methylation, MESH: Chromatin, Evolution, Molecular, [SDV.MHEP.PED] Life Sciences [q-bio]/Human health and pathology/Pediatrics, MESH: Protein Binding, Humans, Protein Interaction Domains and Motifs, Amino Acid Sequence, Cell Nucleus, MESH: Protein Interaction Domains and Motifs, [SDV.MHEP.PED]Life Sciences [q-bio]/Human health and pathology/Pediatrics, MESH: Humans, Research, Nucleolus, Repressor Proteins, MESH: Protein Processing, Post-Translational, MESH: Spumavirus, Spumavirus, lcsh:RC581-607, Protein Processing, Post-Translational, Retroviridae Infections
Abstract: Background Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated. Results We show that the isolated GRI and GRIII boxes act as nucleolar localization signals. In contrast, both the entire Gag CTD and the isolated GRII box, which contains the chromatin-binding motif, target the nucleolus exclusively upon mutation of the evolutionary conserved arginine residue at position 540 (R540), which is a key determinant of FV Gag chromatin tethering. We also provide evidence that Gag localizes in the nucleolus during FV replication and uncovered that the viral protein interacts with and is methylated by Protein Arginine Methyltransferase 1 (PRMT1) in a manner that depends on the R540 residue. Finally, we show that PRMT1 depletion by RNA interference induces the concentration of Gag C-terminus in nucleoli. Conclusion Altogether, our findings suggest that methylation by PRMT1 might finely tune the subnuclear distribution of Gag depending on the stage of the FV replication cycle. The role of this step for viral replication remains an open question. Electronic supplementary material The online version of this article (10.1186/s12977-018-0428-z) contains supplementary material, which is available to authorized users.
Published: 2018

34. Zika virus induces massive cytoplasmic vacuolization and paraptosis‐like death in infected cells

Author: Pierre Génin, Nicoletta Casartelli, Laurent Meertens, Julien Burlaud-Gaillard, Olivier Schwartz, Laura Sinigaglia, Etienne Simon-Loriere, Alex A. Compton, Nicolas Roy, Caroline Demangel, Philippe Roingeard, Nolwenn Jouvenet, Timothée Bruel, Ali Amara, Sonia Amraoui, Françoise Porrot, Arnaud Moris, Blandine Monel, Florence Guivel-Benhassine, Robert Weil, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Pathogénèse, Pathologie cellulaire : aspects moléculaires et viraux / Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS), Génomique virale et vaccination, Immunobiologie de l'Infection - Immunobiology of Infection, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), AC was supported from postdoctoral fellowships from the Pasteur Foundation and ANRS. Work was supported by grants from the 'CHIKV-Viro-Immuno' ANR-14-CE14-0015-01 and 'TIMTAMDEN' ANR-14-CE14-0029 projects, the Labex IBEID program ANR-10-IHUB-0002, the Labex VRI program ANR-10-LABX-77 and Institut Pasteur., We thank members of the Virus & Immunity Unit for discussions. We thank Van-Mai Cao-Lormeau, Institut Louis Malardé (French Polynesia) and Myrielle Dupont-Rouzeyrol (Institut Pasteur Nouvelle Calédonie) for the gift of reagents. We thank Stephane Petres (Institut Pasteur) for preparation of the 4G2 antibody and the Imagopole (Institut Pasteur) for help in image acquisition., ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours
Subjects: 0301 basic medicine, MESH: Cell Death, MESH: Signal Transduction, Vacuole, Endoplasmic Reticulum, Paraptosis, IFITM, Phosphatidylinositol 3-Kinases, 0302 clinical medicine, Cytopathogenic Effect, Viral, Cells, Cultured, Cytopathic effect, ZIKA virus, General Neuroscience, RNA-Binding Proteins, Articles, 3. Good health, Cell biology, cell death, MESH: Epithelial Cells, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Membrane Proteins, MESH: SEC Translocation Channels, Signal Transduction, MESH: Cells, Cultured, Programmed cell death, MESH: Zika Virus, Biology, Virus, General Biochemistry, Genetics and Molecular Biology, cytopathic effect, MESH: Vacuoles, 03 medical and health sciences, MESH: Endoplasmic Reticulum, Humans, Molecular Biology, MESH: Humans, General Immunology and Microbiology, MESH: Proto-Oncogene Proteins c-akt, Endoplasmic reticulum, Membrane Proteins, Epithelial Cells, Fibroblasts, MESH: Cytopathogenic Effect, Viral, Virology, MESH: Astrocytes, 030104 developmental biology, MESH: RNA-Binding Proteins, Viral replication, Vacuolization, MESH: Fibroblasts, MESH: Phosphatidylinositol 3-Kinases, Astrocytes, Vacuoles, paraptosis, Proto-Oncogene Proteins c-akt, SEC Translocation Channels, 030217 neurology & neurosurgery
Abstract: International audience; The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.
Published: 2017

35. Deciphering the role of the chorioallantoic membrane in eggshell decalcification during chicken embryonic development

Author: Halgrain Maeva, Georgeault Sonia, Nelly Bernardet, Hincke, Maxwell T., Sophie Réhault-Godbert, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, University of Ottawa [Ottawa], LE STUDIUM | Institute for advanced studies - Loire Valley, Maxwell Hincke, Sophie Réhault-Godbert, and Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)
Subjects: animal structures, [SDV]Life Sciences [q-bio]
Abstract: International audience; The chicken eggshell (ES) consists of 95% calcium carbonate and 3.5% organic matter and represents the first physical barrier to protect the developing embryo. During the second half of development, calcium ions from the inner ES are progressively solubilized to support mineralization of the embryonic skeleton. This process is mediated by the chorioallantoic membrane (CAM), which is an extraembryonic structure that adheres to the eggshell membrane (ESM) that lines the ES. The CAM surrounds the embryo and egg contents by day 11 of incubation (ED11) and is fully differentiated and functionally active by ED15.We explored the morphological modifications of the ES, ESM and the CAM at these two stages by scanning electron microscopy, and observed that the ESM becomes progressively detached from the inner ES, which is degraded during embryonic development. Concomitantly, the CAM undergoes major structural changes, which include the progressive differentiation of villous cells that extend to reach the ESM and the ES.This study is currently being complemented by transcriptomic analysis of the CAM to identify the genes associated with changes in its structure and with ES demineralization at ED11 and ED15, in order to investigate our hypothesis that the CAM plays a major role in embryo defense (solubilization of ES antimicrobials/recruitment of immune cells) to compensate for eggshell weakening and increased susceptibility to pathogen attack from the external environment.

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35 results on '"Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours)"'

1. The endocytic recycling compartment serves as a viral factory for hepatitis E virus

2. Broadly neutralizing anti-HIV-1 antibodies tether viral particles at the surface of infected cells

3. Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe

4. Structural modifications of the chorioallantoic membrane and the inner eggshell during chicken embryonic development

5. Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate

6. Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress

7. Les usages de Twitter au festival de Cannes. Pratiques numériques et construction des opinions esthétiques

8. 12/111phiA Prophage Domestication Is Associated with Autoaggregation and Increased Ability to Produce Biofilm in Streptococcus agalactiae

9. Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium

10. Combination of β‐(1, 3)‐D‐glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia

11. Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release

12. Non-permissive human conventional CD1c+ dendritic cells enable trans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization

13. Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

14. Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites

15. Emergency department outcome of elderly patients assisted by professional home services, the EPIGER study

16. A molecular determinant of West Nile virus secretion and morphology as a target for viral attenuation

17. BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission

18. Evidence That Zika Virus Is Transmitted by Breastfeeding to Newborn A129 (Ifnar1 Knock-Out) Mice and Is Able to Infect and Cross a Tight Monolayer of Human Intestinal Epithelial Cells

19. Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid

20. A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

21. Differentiation-dependent susceptibility of human muscle cells to Zika virus infection

22. Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

23. A previously unreported frameshift <scp>ATP</scp> 2C1 mutation in a generalized Hailey–Hailey disease

24. The Atlastin ER-shaping proteins facilitate Zika virus replication

25. Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe

26. Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1

27. Ultrasonic identification technique in recycling of lithium ion batteries

28. Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety

29. Oscillating endoskeletal antibubbles

30. Treatment of Pseudomonas aeruginosa Biofilm Present in Endotracheal Tubes by Poly- l -Lysine

31. The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

32. Asymmetric oscillations of endoskeletal antibubbles

33. The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

34. Zika virus induces massive cytoplasmic vacuolization and paraptosis‐like death in infected cells

35. Deciphering the role of the chorioallantoic membrane in eggshell decalcification during chicken embryonic development

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35 results on '"Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours)"'

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