Author: "François-Loïc Cosset" - Searchworks@Jio Institute Digital Library Search Results

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51. The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

Author: Bertrand Boson, Bingjie Zhou, Vincent Legros, Dimitri Lavillette, Cyrille Mathieu, François-Loïc Cosset, Solène Denolly, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Immunobiologie des infections virales – Immunobiology of Viral Infections (IbIV), 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), and 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)
Subjects: 0303 health sciences, Chemistry, viruses, 030302 biochemistry & molecular biology, Mutant, Cell, Golgi apparatus, 3. Good health, Nucleoprotein, Cell biology, 03 medical and health sciences, symbols.namesake, medicine.anatomical_structure, Membrane protein, Cytoplasm, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, symbols, medicine, Secretory pathway, Intracellular, 030304 developmental biology
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely β-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.
Published: 2020

52. Antigen-specific tolerance approach for rheumatoid arthritis: Past, present and future

Author: Audrey Page, Floriane Fusil, and François-Loïc Cosset
Subjects: medicine.medical_treatment, Disease, Autoantigens, Proinflammatory cytokine, Arthritis, Rheumatoid, 03 medical and health sciences, 0302 clinical medicine, Immune system, Rheumatology, medicine, Immune Tolerance, Humans, 030212 general & internal medicine, B cell, Autoantibodies, 030203 arthritis & rheumatology, business.industry, Autoantibody, Immunosuppression, medicine.disease, Clinical trial, medicine.anatomical_structure, Rheumatoid arthritis, Immunology, Cytokines, business
Abstract: Rheumatoid arthritis is a chronic systemic autoimmune disease, affecting mainly the joints. It is caused by an adaptive immune reaction against self-antigens, leading to the over production of inflammatory cytokines and autoantibodies, mainly mediated by autoreactive CD4+ T cells and pathological B cell clones. The treatment options currently available rely on palliative global immunosuppression and do not restore tolerance to self-components. Here, we review antigen-specific tolerance approaches that have been developed to inhibit or delete autoreactive clones, while maintaining a potent immune system for rheumatoid arthritis. The first attempts relied on the oral ingestion of self-reactive peptides, with lukewarm results in human clinical trials. To enhance treatment efficacy, self-peptides have been engineered and combined with immunosuppressive molecules. In addition, several routes of delivery have been tested, in particular, nanoparticles carrying self-antigens and immunomodulatory molecules. More recently, transfer of immune cells, such as tolerogenic dendritic cells or regulatory T cells, has been considered to restore tolerance. Although promising results have been obtained in mouse models, the translation to humans remains highly challenging, mainly because the disease is already well developed when treatments start and because patient's specific self-antigens are often unknown. Nevertheless, these approaches hold great promises for long-term RA treatment.
Published: 2020

53. Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Author: Page, Audrey, Fusil, Floriane, François-Loïc, Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Référent HAL, CIRI, 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), and 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)
Subjects: promoter, [SDV]Life Sciences [q-bio], Genetic Vectors, Lentivirus, transgene, Gene Transfer Techniques, lcsh:QR1-502, Gene Expression, lentiviral vectors, integration, Genetic Therapy, Review, Cellular Reprogramming, lcsh:Microbiology, [SDV] Life Sciences [q-bio], Gene Expression Regulation, Transduction, Genetic, sensor, Animals, Humans, Transgenes, synthetic biology, Promoter Regions, Genetic, induction, signal
Abstract: International audience; Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.
Published: 2020

54. HIV fusion: Catch me if you can

Author: Solène Denolly, François-Loïc Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Référent HAL, CIRI, 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), 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), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Electron Microscope Tomography, [SDV]Life Sciences [q-bio], Cell, Human immunodeficiency virus (HIV), Electrons, HIV Infections, medicine.disease_cause, Biochemistry, Membrane Fusion, Cell Line, 03 medical and health sciences, Viral envelope, medicine, Humans, Editors' Picks, Lipid bilayer, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Fusion, Membrane Glycoproteins, Total internal reflection fluorescence microscope, 030102 biochemistry & molecular biology, Chemistry, Vesicle, Cell Membrane, Cryoelectron Microscopy, env Gene Products, Human Immunodeficiency Virus, Virion, Membrane Proteins, Lipid bilayer fusion, Cell Biology, Cell biology, [SDV] Life Sciences [q-bio], HEK293 Cells, 030104 developmental biology, medicine.anatomical_structure, Microscopy, Fluorescence, Host-Pathogen Interactions, Editors' Picks Highlights, HIV-1, HeLa Cells
Abstract: To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.
Published: 2020

55. Baboon Envelope Pseudotyped 'Nanoblades' Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34

Author: Alejandra, Gutierrez-Guerrero, Maria Jimena, Abrey Recalde, Philippe E, Mangeot, Caroline, Costa, Ornellie, Bernadin, Séverine, Périan, Floriane, Fusil, Gisèle, Froment, Adriana, Martinez-Turtos, Adrien, Krug, Francisco, Martin, Karim, Benabdellah, Emiliano P, Ricci, Simone, Giovannozzi, Rik, Gijsbers, Eduard, Ayuso, François-Loïc, Cosset, and Els, Verhoeyen
Subjects: B cell, gene editing, nanoblade, Genome Editing, T cell, immunotherapy, CRISPR/Cas9, gene therapy, Original Research, hematopoietic stem cells
Abstract: Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized “nanoblades,” a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30–40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.
Published: 2020

56. A longitudinal study of SARS-CoV-2 infected patients shows high correlation between neutralizing antibodies and COVID-19 severity

Author: Guillaume Thierry, Philippe Berthelot, Solène Denolly, Thomas Bourlet, Josselin Rigaill, François-Loïc Cosset, Sylvie Pillet, Manon Vogrig, Carole Pélissier, Elisabeth Botelho-Nevers, Thierry Walzer, Bruno Pozzetto, Bertrand Boson, Omran Allatif, Vincent Legros, Stéphane Paul, Paul O. Verhoeven, Florence Grattard, and Sylvie Gonzalo
Subjects: biology, business.industry, viruses, virus diseases, Disease, Asymptomatic, Pathogenesis, Titer, Immune system, Intensive care, Immunology, biology.protein, Medicine, Antibody, medicine.symptom, business, Neutralizing antibody
Abstract: Understanding the immune responses elicited by SARS-CoV-2 infection is critical in terms of protection from re-infection and, thus, for public health policy and for vaccine development against the COVID-19. Here, using either live SARS-CoV-2 particles or retroviruses pseudotyped with the SARS-CoV-2 S viral surface protein (Spike), we studied the neutralizing antibody (nAb) response in serum specimens from a cohort of 140 SARS-CoV-2 qPCR-confirmed patients, including patient with mild symptoms but also more severe form including those that require intensive care. We show that nAb titers were strongly correlated with disease severity and with anti-Spike IgG levels. Indeed, patients from intensive care units exhibited high nAb titers, whereas patients with milder disease symptoms displayed heterogenous nAb titers and asymptomatic or exclusive outpatient care patients had no or poor nAb levels. We found that the nAb activity in SARS-CoV-2-infected patients displayed a relatively rapid decline after recovery, as compared to individuals infected with alternative coronaviruses. We show the absence of cross-neutralization between endemic coronaviruses and SARS-CoV-2, indicating that previous infection by human coronaviruses may not generate protective nAb against SARS-CoV-2 infection. Finally, we found that the D614G mutation in the Spike protein, which has recently been identified as the major variant now found in Europe, does not allow neutralization escape. Altogether, our results contribute to the understanding of the immune correlate of SARS-CoV-2 induced disease and claim for a rapid evaluation of the role of the humoral response in the pathogenesis of SARS-CoV-2.
Published: 2020

57. Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

Author: Langlois, Anne-Claire, Manzoni, Giulia, Vincensini, Laetitia, Coppée, Romain, Marinach, Carine, Guérin, Maryse, Huby, Thierry, Carrière, Véronique, François-Loïc, Cosset, Dreux, Marlène, Rubinstein, Eric, Silvie, Olivier, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Unité de recherche sur les maladies cardiovasculaires et métaboliques, UMR S 1166, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Sorbonne Université (SU), Hôpital St Antoine, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Virus, mmunité innée et trafic vésiculaire - Vesicular trafficking, innate response and viruses (VIV), Centre de Recherche Saint-Antoine (CR Saint-Antoine), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases [IHU ICAN], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Mère et enfant en milieu tropical : pathogènes, système de santé et transition épidémiologique (MERIT - UMR_D 216), Institut de Recherche pour le Développement (IRD)-Université de Paris (UP), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Trafic Vésiculaire, Réponse Innée et Virus – Vesicular trafficking, Innate response, Mère et enfant en milieu tropical : pathogènes, système de santé et transition épidémiologique (MERIT - UMR_D 261), Institut de Recherche pour le Développement (IRD)-Université Paris Cité (UPCité), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), 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-16-CE15-0004,MALINV,Identification des facteurs de virulence de Plasmodium impliqués dans l'invasion des hépatocytes(2016), and Gestionnaire, Hal Sorbonne Université
Subjects: CD36 Antigens, Models, Molecular, Plasmodium, [SDV]Life Sciences [q-bio], lcsh:R, lcsh:Medicine, Article, Tetraspanin 28, Malaria, Parasite biology, Mice, Protein Domains, Sporozoites, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, parasitic diseases, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Hepatocytes, Animals, Humans, lcsh:Q, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Amino Acid Sequence, lcsh:Science, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract: International audience; Sporozoite forms of the Plasmodium parasite, the causative agent of malaria, are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytes. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.
Published: 2020

58. Massive clonal expansion of polycytotoxic skin and blood CD8

Author: Axel Patrice, Villani, Aurore, Rozieres, Benoît, Bensaid, Klara Kristin, Eriksson, Amandine, Mosnier, Floriane, Albert, Virginie, Mutez, Océane, Brassard, Tugba, Baysal, Mathilde, Tardieu, Omran, Allatif, Floriane, Fusil, Thibault, Andrieu, Denis, Jullien, Valérie, Dubois, Catherine, Giannoli, Henri, Gruffat, Marc, Pallardy, François-Loïc, Cosset, Audrey, Nosbaum, Osami, Kanagawa, Janet L, Maryanski, Daniel, Yerly, Jean-François, Nicolas, and Marc, Vocanson
Subjects: integumentary system, musculoskeletal, neural, and ocular physiology, Immunology, Receptors, Antigen, T-Cell, SciAdv r-articles, macromolecular substances, CD8-Positive T-Lymphocytes, Clone Cells, Immunophenotyping, nervous system, Stevens-Johnson Syndrome, Humans, Prospective Studies, Research Articles, Research Article
Abstract: This study highlights the key role of polycytotoxic CD8+ T cells in the severity of toxic epidermal necrolysis., Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous adverse drug reaction. To better understand why skin symptoms are so severe, we conducted a prospective immunophenotyping study on skin and blood. Mass cytometry results confirmed that effector memory polycytotoxic CD8+ T cells (CTLs) are the main leucocytes in TEN blisters at the acute phase. Deep T cell receptor (TCR) repertoire sequencing identified massive expansion of unique CDR3 clonotypes in blister cells. The same clones were highly expanded in patient’s blood, and the degree of their expansion showed significant correlation with disease severity. By transducing α and β chains of the expanded clonotypes into a TCR-defective cell line, we confirmed that those cells were drug specific. Collectively, these results suggest that the relative clonal expansion and phenotype of skin-recruited CTLs condition the clinical presentation of cutaneous adverse drug reactions.
Published: 2020

59. Preliminary Evidence for Hepatitis Delta Virus Exposure in Patients Who Are Apparently Not Infected With Hepatitis B Virus

Author: Isabelle, Chemin, Flor H, Pujol, Caroline, Scholtès, Carmen L, Loureiro, Fouzia, Amirache, Massimo, Levrero, Fabien, Zoulim, Jimena, Pérez-Vargas, and François-Loïc, Cosset
Subjects: Adult, Aged, 80 and over, Male, Hepatitis B virus, Adolescent, Hepatitis D, Chronic, Hepacivirus, Hepatitis C, Chronic, Middle Aged, Venezuela, Young Adult, Clinical Observations in Hepatology, Hepatitis B, Chronic, Humans, RNA, Viral, Female, Hepatitis Antibodies, Hepatitis Delta Virus, Child, Indigenous Peoples, Massive Hepatic Necrosis, Aged
Published: 2020

60. Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytic cells

Author: Laetitia Vincensini, Véronique Carrière, Giulia Manzoni, Marlène Dreux, Anne-Claire Langlois, Eric Rubinstein, Maryse Guerin, Thierry Huby, François-Loïc Cosset, Carine Marinach, Olivier Silvie, and Romain Coppée
Subjects: Liver infection, Hepatitis C virus, Biology, medicine.disease, biology.organism_classification, medicine.disease_cause, Plasmodium, Virology, Tetraspanin, parasitic diseases, medicine, Scavenger receptor, Receptor, Malaria, CD81
Abstract: Sporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.IMPORTANCEMalaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax. This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.
Published: 2020
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61. Le virus de l’hépatite Delta : Un agent pathogènequi retourne facilement sa veste ?

Author: Anaïs Vignon, Jimena Pérez-Vargas, Solène Denolly, François-Loïc Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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 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), and 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)
Subjects: 0303 health sciences, [SDV]Life Sciences [q-bio], HEPATITIS DELTA, General Medicine, Biology, medicine.disease, Virology, General Biochemistry, Genetics and Molecular Biology, Virus, 3. Good health, 03 medical and health sciences, 0302 clinical medicine, Coinfection, medicine, 030211 gastroenterology & hepatology, Pathogen, 030304 developmental biology
Abstract: International audience; Le virus de l'hépatite Delta Un agent pathogène qui retourne facilement sa veste ? Anaïs Vignon, Solène Denolly, Jimena Perez-Vargas, François-Loïc Cosset > Le virus de l'hépatite Delta (VHD) a été découvert il y a 40 ans dans le foie de personnes infectées par le virus de l'hépatite B (VHB, de la famille des Hepadnaviridae). Parmi les 250 millions de personnes chroniquement infectées par VHB, 15 à 20 millions sont coinfectées par VHD [1]. La surinfection par VHD aggrave l'hépatite déjà causée par VHB, et augmente le risque de développer cirrhose et hépatocarcinome [2]. Du fait de son association avec VHB, VHD est connu en tant que virus « satellite » de VHB, ce qui signifie qu'il en est dépendant (Figure 1) [3]. La particule virale de VHD est formée d'un complexe ribonucléoprotéique constitué du génome viral à ARN associé aux deux formes structurelles de la protéine codée par VHD, S-HDAg et L-HDAg, et repose sur les glycoprotéines de surface de VHB pour l'assemblage des virions, leur enveloppement et leur transmission cellulaire (Figure 1A). Ainsi, pour que VHD puisse former sa particule virale et se disséminer, VHB doit être présent dans la même cellule. Lorsque VHB est absent, VHD reste capable de se répliquer et reste plusieurs semaines dans la cellule, mais il est incapable de se disséminer (Figure 1B) [2]. Cependant, l'idée que VHB est l'unique virus « assistant » utilisé par VHD a récemment été remise en cause. En effet, le génome et les protéines de VHD ont été détectés dans les glandes salivaires de patients atteints du syndrome de Sjögren (une maladie auto-immune systémique), alors que ni les séquences génomiques de VHB ni des anticorps
Published: 2020

62. Structural Basis of Neutralization by Human Antibodies Targeting Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Gc

Author: Laura M. Walker, Dafna M. Abelson, François-Loïc Cosset, Pablo Guardado-Calvo, Natalia Freitas, J. Maximilian Fels, Elisabeth K. Nyakatura, Daniel P. Maurer, Jonathan R. Lai, Jan Hellert, Ariel S. Wirchnianski, Jason S. McLellan, Kartik Chandran, Akaash K. Mishra, Zachary A. Bornholdt, Ahmed Haouz, Olivia Vergnolle, and Félix A. Rey
Subjects: chemistry.chemical_classification, Epitope mapping, biology, chemistry, biology.protein, Antibody, Yeast display, Neutralizing antibody, Glycoprotein, Pathogen, Virology, Crimean Congo hemorrhagic fever virus, Neutralization
Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV), the only endemic biosafety level 4 pathogen in Europe, is the world’s most widely distributed tick-borne zoonotic virus with a 30% fatality rate in humans, underlining the need for specific therapeutics and vaccines. The CCHFV membrane fusion glycoprotein Gc is the main target of the host neutralizing antibody response. Here we describe the structure of pre-fusion Gc in complex with the antigen-binding fragments (Fabs) corresponding to a therapeutically potent bispecific antibody as well as the structure of unbound Gc in its post-fusion conformation. Our findings suggest that one Fab blocks insertion of the fusion loops into the target membrane, whereas the other blocks formation of the post-fusion trimer. Combined with yeast display of mutagenized Gc, the structures allowed epitope mapping of a large panel of neutralizing antibodies. These data provide the essential molecular underpinnings for developing vaccines and therapeutics against CCHFV.
Published: 2020

63. Farnesoid X receptor‐α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo

Author: Christophe Ramière, Floriane Fusil, Vincent Lotteau, Anaïs Ollivier, Camille Ménard, François-Loïc Cosset, Karim Mouzannar, Patrice Andre, and Benoît Lacombe
Subjects: 0301 basic medicine, Hepatitis B virus, Receptors, Cytoplasmic and Nuclear, In Vitro Techniques, Ligands, Virus Replication, medicine.disease_cause, Biochemistry, Virus, Mice, 03 medical and health sciences, 0302 clinical medicine, Proviruses, Genetics, medicine, Animals, Humans, Gene silencing, Molecular Biology, Host factor, Mice, Inbred C3H, Chemistry, Hep G2 Cells, cccDNA, Hepatitis B, Molecular biology, digestive system diseases, HBx, 030104 developmental biology, Gene Expression Regulation, Nuclear receptor, DNA, Viral, Female, Farnesoid X receptor, 030217 neurology & neurosurgery, Biotechnology
Abstract: Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.
Published: 2018

64. Evidence for long-term association of virion-delivered HBV core protein with cccDNA independently of viral protein production

Author: Héloïse Auclair, Julie Lucifora, François-Loïc Cosset, Caroline Pons, Émilie Charles, Florentin Pastor, Anna Salvetti, Floriane Fusil, David Durantel, Michel Rivoire, Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Département cancer environnement (Centre Léon Bérard - Lyon), Centre Léon Bérard [Lyon], Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and 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)
Subjects: HuHep, liver-humanized mice, Viral protein, Short Communication, viruses, rcDNA, relaxed circular DNA, ChIP, HBVΔX, HBx-deficient HBV, RC799-869, Biology, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Internal Medicine, medicine, Protein biosynthesis, Immunology and Allergy, 030304 developmental biology, Hepatitis B virus, H3K27Ac, histone 3 lysine 27 acetylation, 0303 health sciences, CAM, Hepatology, HBc, HBc, HBV core protein, Gastroenterology, virus diseases, cccDNA, Diseases of the digestive system. Gastroenterology, Virology, dHepaRG, differentiated HepaRG cells, digestive system diseases, 3. Good health, ChIP, chromatin immunoprecipitation, HBx, Capsid, FA, formaldehyde, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 030211 gastroenterology & hepatology, cccDNA, covalently closed circular DNA, Hepatitis B Virus, PHHs, primary human hepatocytes, Chromatin immunoprecipitation
Abstract: Background & Aims HBV persists in the nucleus of infected hepatocytes as a covalently closed circular DNA (cccDNA) episome that constitutes the template for viral RNA and protein synthesis. Both HBx and HBc (core) viral proteins associate with cccDNA but, while HBx is required for viral transcription, the role of HBc is still unclear. The aim of this study was to determine if HBc derived from incoming nucleocapsid can associate with cccDNA before the onset of viral transcription and protein production. Methods Chromatin immunoprecipitation assays were performed in native conditions. In addition, differentiated HepaRG (dHepaRG) cells infected with HBx-deficient HBV were used to investigate if HBc delivered by incoming virions can associate with cccDNA. Results Our results indicate that HBc can associate with cccDNA in the absence of viral transcription and de novo protein synthesis. In dHepaRG cells, this association is stable for at least 6 weeks. Conclusion These results suggest that virion-delivered HBc may participate at an early stage of cccDNA formation and/or transcription. Lay summary The hepatitis B virus genome is released into the nucleoplasm of infected cells after disassembly of the viral nucleocapsids at the nuclear membrane. Herein, we show for the first time that virion-delivered hepatitis B core protein, a component of the viral capsid, can stably associate with integrated viral DNA., Graphical abstract, Highlights • The HBV episome (cccDNA) is formed in the nucleus following disassembly of incoming nucleocapsids. • Native ChIP analysis indicates that virion-delivered HBc proteins can stably associate with cccDNA. • Association of virion-delivered HBc does not require de novo viral protein synthesis. • These results suggest that virion-delivered HBc may participate in cccDNA formation and/or transcriptional regulation.
Published: 2021

65. Report of One-Year Prospective Surveillance of SARS-CoV-2 in Dogs and Cats in France with Various Exposure Risks: Confirmation of a Low Prevalence of Shedding, Detection and Complete Sequencing of an Alpha Variant in a Cat

Author: Solène Denolly, Meriadeg Ar Gouilh, Matthieu Fritz, Bryce Leterrier, Emilie Krafft, François-Loïc Cosset, Angeli Kodjo, Sophie Levaltier, Sophie Angelloz-Pessey, Eric M. Leroy, Nicolas Nesi, Sandrine Corbet, Bertrand Boson, and Vincent Legros
Subjects: Male, medicine.medical_specialty, prevalence, Physiology, Context (language use), Cat Diseases, Real-Time Polymerase Chain Reaction, Microbiology, Asymptomatic, Article, Serology, Dogs, Virology, Epidemiology, medicine, Animals, Humans, Dog Diseases, Prospective Studies, Viral shedding, skin and connective tissue diseases, variants, CATS, Sequence Analysis, RNA, SARS-CoV-2, Transmission (medicine), business.industry, fungi, Zoonosis, COVID-19, Pets, sequencing, zoonosis, medicine.disease, QR1-502, Virus Shedding, body regions, Infectious Diseases, Cats, RNA, Viral, France, medicine.symptom, business
Abstract: Despite the probable zoonotic origin of SARS-CoV-2, only limited research efforts have been made to understand the role of companion animals in SARS-CoV-2 epidemiology. According to recent serological prevalence studies, human-to-companion animal transmission is quite frequent, which led us to consider that the risk of SARS-CoV-2 transmission from animal to human, albeit negligible in the present context, may have been underestimated. In this study, we provide the results of a prospective survey that was conducted to evaluate the SARS-CoV-2 isolation rate by qRT-PCR in dogs and cats with different exposure risks and clinical statuses. From April 2020 to April 2021, we analyzed 367 samples and investigated the presence of SARS-CoV-2 RNA using qRT-PCR. Only four animals tested positive, all of them being cats. Three cats were asymptomatic and one presented a coryza-like syndrome. We describe in detail the infection in two cats and the associated clinical characteristics. Importantly, we obtained SARS-CoV-2 genomes from one infected animal and characterized them as Alpha variants. This represents the first identification of the SARS-CoV-2 Alpha variant in an infected animal in France.
Published: 2021

66. Sensing of cell-associated HTLV by plasmacytoid dendritic cells is regulated by dense β-galactoside glycosylation

Author: Assil, Sonia, Futsch, Nicolas, Décembre, Elodie, Alais, Sandrine, Gessain, Antoine, François-Loïc, Cosset, Mahieux, Renaud, Dreux, Marlène, Dutartre, Hélène, Bodescot, Myriam, Dynamiques eco-évolutives des maladies infectieuses - - ECOFECT2011 - ANR-11-LABX-0048 - LABX - VALID, 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 (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), This work was supported by 'Ligue Nationale contre le Cancer, équipe labélisée program' EL2013-3Mahieux to Dr Hélène Dutartre and Pr Renaud Mahieux, by ‘‘Agence Nationale pour la Recherche’’ (ANR-JCJC-EXAMIN), ‘‘Agence Nationale pour la Recherche contre le SIDA et les Hépatites Virales’’ (ANRS-AO 2016-01) and LabEx Ecofect (ANR-11-LABX-0048) of the 'Université de Lyon', within the program 'Investissements d’Avenir' (ANR-11-IDEX-0007) to Dr Marlène Dreux, and by LabEx Ecofect (ANR-11-LABX-0048) of the 'Université de Lyon', within the program 'Investissements d’Avenir' (ANR-11-IDEX-0007) to Dr François-Loïc Cosset. Sonia Assil’s PhD fellowship was sponsored by the French ministry and the 'Ligue contre le Cancer'. Nicolas Futsch’s PhD fellowship was sponsored by the 'Ligue contre le Cancer'., ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), 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 Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: RNA viruses, Glycosylation, viruses, T-Lymphocytes, Glycobiology, Pathology and Laboratory Medicine, Biochemistry, Jurkat Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Lectins, Medicine and Health Sciences, Biology (General), Post-Translational Modification, Staining, Human T-lymphotropic virus 1, Human T-lymphotropic virus 2, Cell Staining, Drugs, hemic and immune systems, Medical Microbiology, Viral Pathogens, Interferon Type I, Viruses, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.IMM]Life Sciences [q-bio]/Immunology, Cytokines, Pathogens, Research Article, Cell Binding, Cell Physiology, [SDV.IMM] Life Sciences [q-bio]/Immunology, QH301-705.5, Viral Structure, Research and Analysis Methods, Microbiology, Virology, Retroviruses, Humans, Microbial Pathogens, Pharmacology, Biology and life sciences, Heparin, Organisms, Interferon-alpha, Proteins, Galactosides, Dendritic Cells, Htlv-1, Cell Biology, RC581-607, HTLV-I Infections, Immunity, Innate, Specimen Preparation and Treatment, Immunologic diseases. Allergy, Viral Transmission and Infection
Abstract: Human T Lymphotropic virus (HTLV) infection can persist in individuals resulting, at least in part, from viral escape of the innate immunity, including inhibition of type I interferon response in infected T-cells. Plasmacytoid dendritic cells (pDCs) are known to bypass viral escape by their robust type I interferon production. Here, we demonstrated that pDCs produce type I interferons upon physical cell contact with HTLV-infected cells, yet pDC activation inversely correlates with the ability of the HTLV-producing cells to transmit infection. We show that pDCs sense surface associated-HTLV present with glycan-rich structure referred to as biofilm-like structure, which thus represents a newly described viral structure triggering the antiviral response by pDCs. Consistently, heparan sulfate proteoglycans and especially the cell surface pattern of terminal β-galactoside glycosylation, modulate the transmission of the immunostimulatory RNA to pDCs. Altogether, our results uncover a function of virus-containing cell surface-associated glycosylated structures in the activation of innate immunity., Author summary Human T Lymphotropic virus type (HTLV) establishes persistent infections, leading to adult T-cell Leukemia, a life-threatening cancer in chronically-infected individuals. Viral persistence likely results from a failure of immune responses to eradicate viral replication, a least in part, by viral escape from innate immunity, and notably via decreased production of type I interferons (IFN-I) by infected cells. Plasmacytoid dendritic cells (pDCs) are known as robust producers of IFN-I in response to virus stimulation, thus bypassing the viral mechanisms to evade pathogen-sensing pathways in infected cells. However, HTLV particles are not detected in biological fluids of infected individuals, raising the question of the pDC-activating signal. Here, we demonstrate that pDCs produce IFN-I upon physical contacts with HTLV-infected cells. We show that pDCs sense surface associated-HTLV present with glycan-rich structure, referred to as HTLV-biofilm-like structure. Importantly, the sensing of infected cells by pDCs is modulated by the glycosylation pattern at the surface of infected cells. This newly ascribed regulation of innate immunity activation by cell surface-associated glycans might contribute to the differential activation levels of antiviral response to infected cells when their glycosylation profile is modified, such as for chronically infected cells or tumor cells.
Published: 2018

67. Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector

Author: Thomas F. Baumert, Xavier Marniquet, Pierre Cortez, Laurent Mailly, Anna Salvetti, François-Loïc Cosset, Fabien Zoulim, Aurore Inchauspé, Barbara Testoni, Massimo Levrero, Julie Lucifora, David Durantel, Floriane Fusil, Maud Michelet, C Challier, Marie-Louise Michel, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [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), Sanofi-Aventis R&D, SANOFI Recherche, Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Biologie des virus entériques (BVE), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pôle Hépato- digestif, Les Hôpitaux Universitaires de Strasbourg (HUS), Hospices Civils de Lyon (HCL), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), 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), Davoine, Laure-Hélène, 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), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]
Subjects: 0301 basic medicine, medicine.disease_cause, Polymerase Chain Reaction, law.invention, Mice, chemistry.chemical_compound, Adeno-associated virus, Transduction, Genetic, Transcription (biology), law, Viral, Southern, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Blotting, virus diseases, cccDNA, Dependovirus, Hepatitis B, Immune-therapeutics, 3. Good health, Blotting, Southern, Liver, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Recombinant DNA, [SDV.IMM]Life Sciences [q-bio]/Immunology, DNA, Circular, adeno-associated virus, cccdna, hepatitis b virus, immune-competent mouse, immune-therapeutics, animals, blotting, southern, dna replication, dna, circular, dna, viral, dependovirus, disease models, animal, hepatitis b, hepatocytes, liver, mice, plasmids, polymerase chain reaction, transduction, genetic, genetic vectors, Plasmids, DNA Replication, Hepatitis B virus, [SDV.IMM] Life Sciences [q-bio]/Immunology, Genetic Vectors, Circular, Circular DNA, Biology, Transduction, 03 medical and health sciences, Genetic, Viral life cycle, Virology, medicine, Animals, Southern blot, Pharmacology, Animal, DNA, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Molecular biology, digestive system diseases, Disease Models, Animal, 030104 developmental biology, Immune-competent mouse, chemistry, DNA, Viral, Disease Models, Hepatocytes, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: International audience; Hepatitis B Virus (HBV) persists in infected hepatocytes as an episomal covalently-closed-circular DNA mini-chromosome, called cccDNA. As the main nuclear transcription template, HBV cccDNA is a key replication intermediate in the viral life cycle. Little is known about the mechanisms involved in its formation, maintenance and fate under antiviral therapies. This is mainly due to the lack of small immune-competent animal models able to recapitulate the entire HBV replication cycle, including formation of HBV cccDNA. Here we report that HBV cccDNA can be detected by Southern blot analyses in the liver of C57BL6 mice transduced with AAV-HBV. HBV cccDNA persists in the liver of these animals together with the AAV-HBV episome. We also set up a PCR strategy to distinguish the HBV cccDNA from the AAV-HBV episome. These suggest that the AAV-HBV/mouse model might be relevant to test drugs targeting HBV cccDNA regulation and persistence.
Published: 2017

68. Gene Therapy in Fanconi Anemia: A Matter of Time, Safety and Gene Transfer Tool Efficiency

Author: Camille Lévy, Francisco J Roman-Rodriguez, Paula Río, François-Loïc Cosset, Els Verhoeyen, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Centro de Investigaciones Energéticas Medioambientales y Tecnológicas [Madrid] (CIEMAT), 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Genetic enhancement, Genetic Vectors, Hematopoietic stem cell, Viral vector, 03 medical and health sciences, Gene therapy, Fanconi anemia, Drug Discovery, Genetics, medicine, Animals, Humans, Molecular Biology, Genetics (clinical), Targeted cell transduction, business.industry, Lentivirus, Gene Transfer Techniques, Hematopoietic Stem Cell Transplantation, Bone marrow failure, Genetic Therapy, Hematopoietic Stem Cells, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Haematopoiesis, 030104 developmental biology, medicine.anatomical_structure, Pseudotyping, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Immunology, Cancer research, [SDV.IMM]Life Sciences [q-bio]/Immunology, Molecular Medicine, Lentiviral vector, Bone marrow, Stem cell, Reactive oxygen species, business
Abstract: International audience; Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive marrow failure. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential cure since it is a monogenetic disease with mutations in the FANC genes, coding for DNA repair enzymes [1]. However, the collection of hCD34+-cells in FA patients implies particular challenges because of the reduced numbers of progenitor cells present in their bone marrow (BM) [2] or mobilized peripheral blood [3-5]. In addition, the FA genetic defect fragilizes the HSCs [6]. These particular features might explain why the first clinical trials using murine leukemia virus derived retroviral vectors conducted for FA failed to show engraftment of corrected cells. The gene therapy field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent succesful clinical trials for the treatment of patients suffering from adrenoleukodystrophy (ALD) [7], β-thalassemia [8], metachromatic leukodystrophy [9] and Wiskott-Aldrich syndrome [10]. LV trials for X-linked severe combined immunodificiency and Fanconi anemia (FA) defects were recently initiated [11, 12]. Fifteen years of preclinical studies using different FA mouse models and in vitro research allowed us to find the weak points in the in vitro culture and transduction conditions, which most probably led to the initial failure of FA HSC gene therapy. In this review, we will focus on the different obstacles, unique to FA gene therapy, and how they have been overcome through the development of optimized protocols for FA HSC culture and transduction and the engineering of new gene transfer tools for FA HSCs. These combined advances in the field hopefully will allow the correction of the FA hematological defect in the near future.
Published: 2017

69. Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2

Author: Joel E. Schechter, Lishan Su, Chia-Lin Chen, Keigo Machida, François-Loïc Cosset, Jeffrey Y. Huang, Takaji Wakita, Jae U. Jung, Yasuteru Kondo, Chun-Hsiang Wang, Stanley M. Tahara, Lin Zhou, Michael M. C. Lai, Keck School of Medicine [Los Angeles], University of Southern California (USC), Department of Biology - Carolina Center for Genome Sciences - School of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC)-University of North Carolina System (UNC), Academia Sinica, National Institute of Infectious Diseases [Tokyo], Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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 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), and 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)
Subjects: 0301 basic medicine, Co-receptor, Interferon-Induced Helicase, IFIH1, General Physics and Astronomy, Hepacivirus, medicine.disease_cause, Virus Replication, Viral Envelope Proteins, RNA, Small Interfering, Receptors, Immunologic, B-Lymphocytes, Multidisciplinary, virus diseases, Hep G2 Cells, 3. Good health, Host-Pathogen Interactions, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, DEAD Box Protein 58, [SDV.IMM]Life Sciences [q-bio]/Immunology, Clone (B-cell biology), Protein Binding, Signal Transduction, Hepatitis C virus, Science, Biology, General Biochemistry, Genetics and Molecular Biology, Virus, Article, 03 medical and health sciences, Immune system, Viral envelope, Cell Line, Tumor, medicine, Gene silencing, Humans, Gene Library, General Chemistry, Virology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, digestive system diseases, MicroRNAs, Viral Tropism, 030104 developmental biology, HEK293 Cells, Viral replication, Gene Expression Regulation, Immunology, B7-2 Antigen, Immunologic Memory
Abstract: B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts., Infection of B cells by hepatitis C virus (HCV) is poorly understood, but is thought to result in lymphoproliferative disorders. Here, Chen et al. identify CD86 as co-receptor for lymphotropic HCV and show that HCV infection inhibits memory B-cell function.
Published: 2017

70. Gene-corrected human Munc13-4–deficient CD8+ T cells can efficiently restrict EBV-driven lymphoproliferation in immunodeficient mice

Author: Geneviève de Saint Basile, Persis Amrolia, Amandine Durand, Julie Rivière, François-Loïc Cosset, Ida Ricciardelli, Chantal Lagresle-Peyrou, Isabelle André-Schmutz, Tayebeh Soheili, Marina Cavazzana, Anne-Céline Derrien, and Els Verhoeyen
Subjects: 0301 basic medicine, Epstein-Barr Virus Infections, Herpesvirus 4, Human, Immunology, Lymphoproliferative disorders, Mice, SCID, CD8-Positive T-Lymphocytes, Biochemistry, Lymphohistiocytosis, Hemophagocytic, Mice, 03 medical and health sciences, Transduction, Genetic, Animals, Humans, Medicine, Cytotoxic T cell, Gene, Epstein–Barr virus infection, business.industry, Membrane Proteins, FHL3, Genetic Therapy, Cell Biology, Hematology, Familial Hemophagocytic Lymphohistiocytosis, medicine.disease, Lymphoproliferative Disorders, 030104 developmental biology, Membrane protein, UNC13D Gene, business
Abstract: To the editor: Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a hyperinflammatory disease caused by mutations in the UNC13D gene (coding for the Munc13-4 protein); FHL3 accounts for 30% to 35% of FHL cases.[1][1] In cytotoxic T cells (CTLs) and natural killer cells, Munc13-4 helps to
Published: 2016

71. Maturation extracellulaire du virus de l’hépatite C

Author: Solène Denolly, Marion Chanut, François-Loïc Cosset, Christelle Granier, Children's Hospital, Laboratoire d'aérologie (LAERO), Centre National de la Recherche Scientifique (CNRS)-Observatoire Midi-Pyrénées (OMP), Météo France-Centre National d'Études Spatiales [Toulouse] (CNES)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Météo France-Centre National d'Études Spatiales [Toulouse] (CNES)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, NOAA Earth System Research Laboratory (ESRL), National Oceanic and Atmospheric Administration (NOAA), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Observatoire Midi-Pyrénées (OMP), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Météo-France -Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Météo-France -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), and 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)
Subjects: 0303 health sciences, Invisibility, biology, Chemistry, Hepatitis C virus, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, Cloak, General Medicine, medicine.disease_cause, Virology, General Biochemistry, Genetics and Molecular Biology, 3. Good health, 03 medical and health sciences, biology.protein, Extracellular, medicine, Antibody, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: International audience
Published: 2019

72. Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency

Author: David Fenard, Ornellie Bernadin, Anais Girard-Gagnepain, Camille Lévy, Chantal Lagresle-Peyrou, Kuiying Ma, Rana Mhaidly, Hanem Sadek, Marina Cavazzana, Fouzia Amirache, Caroline Costa, Ranjita Devi Moirangthem, Isabelle André, François-Loïc Cosset, Els Verhoeyen, Christian Reimann, Didier Nègre, Agata Cieslak, Vahid Asnafi, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Human Lymphohematopoiesis Laboratory (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lucerne Cantonal Hospital [Luzern, Switzerland], Généthon, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Clinical Investigation Center in Biotherapy, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Human Virology Department, Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Developpement Normal et Pathologique du Système Immunitaire, Université Nice Sophia Antipolis (UNS), Département de Biothérapie [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Université Paris Descartes - Paris 5 (UPD5)-CHU Necker - Enfants Malades [AP-HP], Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)
Subjects: 0301 basic medicine, medicine.medical_treatment, Genetic enhancement, [SDV]Life Sciences [q-bio], CD34, chemical and pharmacologic phenomena, Mice, SCID, Hematopoietic stem cell transplantation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, complex mixtures, Mice, 03 medical and health sciences, 0302 clinical medicine, Mice, Inbred NOD, medicine, Animals, Progenitor cell, ComputingMilieux_MISCELLANEOUS, Severe combined immunodeficiency, integumentary system, Stem Cells, Lentivirus, hemic and immune systems, Gene Therapy, Hematology, bacterial infections and mycoses, medicine.disease, T cell deficiency, 3. Good health, Cell biology, Haematopoiesis, 030104 developmental biology, 030220 oncology & carcinogenesis, Stem cell, Papio
Abstract: T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7–stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC−/− engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC−/− recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.
Published: 2019

73. Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes

Author: Evarist Planet, Nathalie Brandenberg, Marco Cassano, Didier Trono, François-Loïc Cosset, Jimena Pérez-Vargas, Barbara E. Wildhaber, Floriane Fusil, Matthias P. Lutolf, Marc Friedli, and Carmen Unzu
Subjects: Male, 0301 basic medicine, Liver cytology, Primary Cell Culture, Cell Culture Techniques, Biology, ddc:616.07, law.invention, 03 medical and health sciences, 0302 clinical medicine, Liver Biology/Pathobiology, Mice, Inbred NOD, law, Animals, Progenitor cell, Induced pluripotent stem cell, ddc:618, Hepatology, Stem Cells, Bioartificial liver device, Original Articles, Liver regeneration, Culture Media, 3. Good health, Cell biology, 030104 developmental biology, Liver, Cell culture, Case-Control Studies, Hepatocytes, Original Article, 030211 gastroenterology & hepatology, Stem cell, Ex vivo
Abstract: The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.
Published: 2019

74. Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing

Author: Thomas Schaser, Anne Galy, Pia Sträßer, Ornellie Bernadin, Katharina Drechsel, Alejandra Gutierrez-Guerrero, Ian C.D. Johnston, Mirella Mormin, Els Verhoeyen, Andrew Kaiser, François-Loïc Cosset, Nicole Cordes, Constanze Radek, Rita Pfeifer, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, 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), and École Pratique des Hautes Études (EPHE)
Subjects: T cell, medicine.medical_treatment, T-Lymphocytes, [SDV]Life Sciences [q-bio], Genetic Vectors, Endogenous retrovirus, Biology, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Antigen, Viral Envelope Proteins, automated manufacturing, Transduction, Genetic, Genetics, medicine, Animals, Humans, Molecular Biology, Research Articles, pseudotyping, 030304 developmental biology, Glycoproteins, 0303 health sciences, B-Lymphocytes, Lentivirus, Hematopoietic stem cell, Immunotherapy, Genetic Therapy, Antigens, CD20, Hematopoietic Stem Cells, transduction, 3. Good health, Cell biology, CliniMACS Prodigy, medicine.anatomical_structure, Retroviridae, Measles virus, 030220 oncology & carcinogenesis, Leukemia Virus, Gibbon Ape, Pseudotyping, Molecular Medicine, CD34, immunotherapy, Gammaretrovirus, Peptides
Abstract: International audience; Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1((R)), to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.
Published: 2019

75. Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo

Author: Pérez-Vargas, Jimena, Amirache, Fouzia, Boson, Bertrand, Mialon, Chloé, Freitas, Natalia, Sureau, Camille, Fusil, Floriane, François-Loïc, Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université des Antilles (UA), sureau, camille, 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 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), and 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)
Subjects: Science, viruses, Primary Cell Culture, Mice, Transgenic, Hepacivirus, Mice, Viral Envelope Proteins, Mice, Inbred NOD, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Cell Line, Tumor, Animals, Humans, lcsh:Science, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Coinfection, Flavivirus, Virus Assembly, Virion, virus diseases, Vesiculovirus, biochemical phenomena, metabolism, and nutrition, Hepatitis D, digestive system diseases, Ribonucleoproteins, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hepatocytes, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, RNA, Viral, lcsh:Q, Hepatitis Delta Virus
Abstract: International audience; Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleo-protein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/ or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.
Published: 2019

76. Overview of HCV Life Cycle with a Special Focus on Current and Possible Future Antiviral Targets

Author: Bertrand Boson, François-Loïc Cosset, Nathalie Alazard-Dany, Solène Denolly, 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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), 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 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)
Subjects: 0301 basic medicine, medicine.medical_specialty, lcsh:QR1-502, Review, Hepacivirus, DIRECT ACTING ANTIVIRALS, Antiviral Agents, lcsh:Microbiology, 03 medical and health sciences, Global population, 0302 clinical medicine, Virology, Medicine, Humans, Protease Inhibitors, Molecular Targeted Therapy, Intensive care medicine, DAA, Focus (computing), business.industry, Hepatitis C, medicine.disease, 3. Good health, 030104 developmental biology, Infectious Diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HCV, 030211 gastroenterology & hepatology, business, antiviral targets
Abstract: International audience; Hepatitis C infection is the leading cause of liver diseases worldwide and a major health concern that affects an estimated 3% of the global population. Novel therapies available since 2014 and 2017 are very efficient and the WHO considers HCV eradication possible by the year 2030. These treatments are based on the so-called direct acting antivirals (DAAs) that have been developed through research efforts by academia and industry since the 1990s. After a brief overview of the HCV life cycle, we describe here the functions of the different targets of current DAAs, the mode of action of these DAAs and potential future inhibitors.
Published: 2019

77. Maturation extracellulaire du virus de l’hépatite C

Author: Chanut, Marion, Granier, Christelle, François-Loïc, Cosset, Denolly, Solène, Sciences, EDP, Children's Hospital, Laboratoire d'aérologie (LAERO), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Observatoire Midi-Pyrénées (OMP), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Météo-France -Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Météo-France -Centre National de la Recherche Scientifique (CNRS), NOAA Earth System Research Laboratory (ESRL), National Oceanic and Atmospheric Administration (NOAA), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2019

78. Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells

Author: Solène, Denolly, François-Loïc, Cosset, and Natalia, Freitas
Subjects: Cell Culture Techniques, Virion, Hepacivirus, Virus Internalization, Transfection, Antiviral Agents, Hepatitis C, Membrane Fusion, Cell Line, HEK293 Cells, Host-Pathogen Interactions, Liposomes, Humans, Glycoproteins
Abstract: The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.
Published: 2018

79. Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells

Author: François-Loïc Cosset, Solène Denolly, and Natalia Freitas
Subjects: 0301 basic medicine, chemistry.chemical_classification, Liposome, Fusion, Chemistry, Hepatitis C virus, Cell, Lipid bilayer fusion, medicine.disease_cause, Cell biology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Membrane, medicine.anatomical_structure, Viral Receptor, medicine, 030211 gastroenterology & hepatology, Glycoprotein
Abstract: The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.
Published: 2018

80. Evolution of Hepatitis B Virus Receptor NTCP Reveals Differential Pathogenicities and Species Specificities of Hepadnaviruses in Primates, Rodents, and Bats

Author: Jean-Baptiste Pons, Dominique Pontier, François-Loïc Cosset, Lucie Etienne, Barthélémy Ngoubangoye, Stéphanie Jacquet, Corinne Régis, Ariel de Bernardo, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherches Médicales de Franceville (CIRMF), Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Recherches Translationnelles sur le VIH et les maladies infectieuses endémiques er émergentes (TransVIHMI), Institut de Recherche pour le Développement (IRD)-Université de Yaoundé I-Université Cheikh Anta Diop [Dakar, Sénégal] (UCAD)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Département écologie évolutive [LBBE], Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), mulet-marquis, odile, Université Cheikh Anta Diop [Dakar, Sénégal] (UCAD)-Institut de Recherche pour le Développement (IRD)-Université de Yaoundé I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), École normale supérieure de Lyon (ENS de Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), and Recherches Translationnelles sur le VIH et les maladies infectieuses endémiques et émergentes (TransVIHMI)
Subjects: Primates, Hepatitis B virus, Rodent, Immunology, Organic Anion Transporters, Sodium-Dependent, Rodentia, medicine.disease_cause, Microbiology, Host Specificity, Evolution, Molecular, 03 medical and health sciences, 0302 clinical medicine, Orthohepadnavirus, Viral envelope, Species Specificity, Viral Envelope Proteins, biology.animal, Chiroptera, Virology, medicine, [SDV.BID.EVO] Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], Animals, Humans, Primate, Receptor, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Genetics, 0303 health sciences, biology, Symporters, Host (biology), [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], Genetic Variation, [SDV.EE.IEO] Life Sciences [q-bio]/Ecology, environment/Symbiosis, Virus Internalization, biology.organism_classification, Hepatitis B, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Genetic Diversity and Evolution, Insect Science, Hepadnavirus, 030217 neurology & neurosurgery, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract: Human hepatitis B virus (HBV) is a global health problem, affecting more than 250 million people worldwide. HBV-like viruses, named orthohepadnaviruses, also naturally infect nonhuman primates, rodents, and bats, but their pathogenicity and evolutionary history are unclear. Here, we determined the evolutionary history of the HBV receptors NTCP and GPC5 over millions of years of primate, rodent, and bat evolution. We use this as a proxy to understand the pathogenicity of orthohepadnaviruses in mammalian hosts and to determine the implications for species specificity. We found that NTCP, but not GPC5, has evolved under positive selection in primates (27 species), rodents (18 species), and bats (21 species) although at distinct residues. Notably, the positively selected codons map to the HBV-binding sites in primate NTCP, suggesting past genetic “arms races” with pathogenic orthohepadnaviruses. In rodents, the positively selected codons fall outside and within the presumed HBV-binding sites, which may contribute to the restricted circulation of rodent orthohepadnaviruses. In contrast, the presumed HBV-binding motifs in bat NTCP are conserved, and none of the positively selected codons map to this region. This suggests that orthohepadnaviruses may bind to different surfaces in bat NTCP. Alternatively, the patterns may reflect adaptive changes associated with metabolism rather than pathogens. Overall, our findings further point to NTCP as a naturally occurring genetic barrier for cross-species transmissions in primates, which may contribute to the narrow host range of HBV. In contrast, this constraint seems less important in bats, which may correspond to greater orthohepadnavirus circulation and diversity. IMPORTANCE Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. Mammalian HBV-like viruses are also found in nonhuman primates, rodents, and bats. As for most viruses, HBV requires a successful interaction with a host receptor for replication. Cellular receptors are thus key determinants of host susceptibility as well as specificity. One hallmark of pathogenic virus-host relationships is the reciprocal evolution of host receptor and viral envelope proteins, as a result of their antagonistic interaction over time. The dynamics of these so-called “evolutionary arms races” can leave signatures of adaptive selection, which in turn reveal the evolutionary history of the virus-host interaction as well as viral pathogenicity and the genetic determinants of species specificity. Here, we show how HBV-like viruses have shaped the evolutionary history of their mammalian host receptor, as a result of their ancient pathogenicity, and decipher the genetic determinants of cross-species transmissions.
Published: 2018
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81. Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction

Author: Robert E. Lanford, Caroline Marnata, Dimitri Lavillette, François-Loïc Cosset, Lisette Cohen, Célia Boukadida, Dimitri Mompelat, Lucile Warter, Ieva Vasiliauskaite, Judith Fresquet, Aure Saulnier, Félix A. Rey, Peter Karayiannis, Thomas Krey, Nicolas Escriou, Annette Martin, Nicola J. Rose, Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), LabEx ECOFECT Inserm U1111, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génomique virale et vaccination, Southwest National Primate Research Center, Texas Biomedical Research Institute, Department of Medicine, Imperial College London, National Institute for Biological Standards and Control (NIBSC), Medicines and Healthcare Products Regulatory Agency (MHRA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris], Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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), Virologie Structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: viruses, Hepacivirus, Fluorescent Antibody Technique, Simian, medicine.disease_cause, Models, Serial passage, biology, virus diseases, Virus-Cell Interactions, 3. Good health, Models, Animal, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Sequence Analysis, Plasmids, Primates, Hepatitis C virus, Immunoblotting, Molecular Sequence Data, Immunology, Enzyme-Linked Immunosorbent Assay, Viremia, Microbiology, GB virus B, Host Specificity, Virus, Virology, medicine, Animals, Humans, DNA Primers, Life Cycle Stages, NS3, Base Sequence, Animal, Sequence Analysis, DNA, DNA, Virus Internalization, biology.organism_classification, medicine.disease, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, digestive system diseases, HEK293 Cells, Insect Science, Hepatocytes, CD81
Abstract: Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.
Published: 2015

82. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status

Author: François-Loïc Cosset, Thierry Defrance, Fouzia Amirache, Dimitri Lavillette, Justin B. Robbins, Jimmy Mancip, Els Verhoeyen, Sara Calattini, Caroline Costa, Floriane Fusil, Florian Douam, Mansun Law, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), LabEx ECOFECT Inserm U1111, The Scripps Research Institute [La Jolla, San Diego], Lymphocytes B effecteurs et à mémoire – Effector and memory B cells, Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), 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), The Scripps Research Institute [La Jolla], University of California [San Diego] (UC San Diego), University of California-University of California, Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Adoptive cell transfer, Hepacivirus, Plasma cell, Lymphocyte Activation, Mice, Transduction (genetics), Viral Envelope Proteins, Mice, Inbred NOD, Transduction, Genetic, Receptors, Drug Discovery, Neutralizing antibody, Neutralizing, chemistry.chemical_classification, B-Lymphocytes, Tumor, Membrane Glycoproteins, biology, 3. Good health, Cell biology, Protein Transport, medicine.anatomical_structure, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Molecular Medicine, Original Article, Antibody, IgG, Genetic Vectors, Antibodies, Cell Line, Viral vector, Transduction, Genetic, Cell Line, Tumor, Genetics, medicine, Animals, Humans, Molecular Biology, Pharmacology, Receptors, IgG, Lentivirus, Antibody-Dependent Cell Cytotoxicity, Antibodies, Neutralizing, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Virology, HEK293 Cells, chemistry, Immunoglobulin G, biology.protein, Inbred NOD, Glycoprotein, Ex vivo
Abstract: International audience; The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.
Published: 2015

83. A master regulator of tight junctions involved in hepatitis C virus entry and pathogenesis

Author: Solène Denolly, François-Loïc Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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 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), and 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)
Subjects: 0301 basic medicine, Hepatitis C virus, Hepacivirus, Biology, medicine.disease_cause, Tight Junctions, Pathogenesis, 03 medical and health sciences, Occludin, Claudin-1, medicine, Humans, ComputingMilieux_MISCELLANEOUS, Hepatology, Tight junction, Master regulator, Virus Internalization, Cadherins, Hepatitis C, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Virology, Cell biology, 030104 developmental biology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hepatocytes, [SDV.IMM]Life Sciences [q-bio]/Immunology
Abstract: International audience
Published: 2017

84. A serum protein factor mediates maturation and apoB-association of HCV particles in the extracellular milieu

Author: François-Loïc Cosset, Christelle Granier, Thomas Bourlet, Maryse Guerin, Nelly Fontaine, Bruno Pozzetto, Solène Denolly, 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), Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), Université Jean Monnet [Saint-Étienne] (UJM), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), 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), Université Jean Monnet - Saint-Étienne (UJM), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)
Subjects: 0301 basic medicine, Apolipoprotein B, Hepatitis C virus, [SDV]Life Sciences [q-bio], Cholesterol, VLDL, Serum Albumin, Human, Hepacivirus, medicine.disease_cause, Virus, Culture Media, Serum-Free, Microbiology, 03 medical and health sciences, Viral Proteins, 0302 clinical medicine, Apolipoproteins E, Viral Envelope Proteins, Cell Line, Tumor, Extracellular, medicine, Humans, Hepatology, biology, Chemistry, Virus Assembly, Cholesterol, HDL, Albumin, Virion, Extracellular Fluid, Cholesterol, LDL, Hepatitis C, In vitro, 3. Good health, Hypervariable region, 030104 developmental biology, Culture Media, Conditioned, Apolipoprotein B-100, biology.protein, Hepatocytes, 030211 gastroenterology & hepatology, Lipoprotein
Abstract: In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera.We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins.Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (1.08) or very-low density (1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin.The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients.Hepatitis C virus (HCV) particles may associate with apoB and acquire neutral lipids after exiting cells, giving them low-buoyant density. The hypervariable region 1 (HVR1) is a majorviral determinant of E2 that controls this process. Besides lipoproteins, specific serum factors including albumin promote extracellular maturation of HCV virions. HCV particle production in vitro, with media of defined serum conditions, enables production of infectious particles resembling those of chronically infected patients.
Published: 2018

85. A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism

Author: Laurent Mailly, Emilie Carlot, Gabriela Hrebikova, François-Loïc Cosset, Ke Xu, Qiang Ding, Thomas Baumert, Dimitri Lavillette, Alessandra Carbone, Jimmy Mancip, Els Verhoeyen, Loïc Schwaller, Margot Enguehard, Carine Maisse, Linda Dib, Alexander Ploss, Florian Douam, Floriane Fusil, Roland Montserret, Francesca Nadalin, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Department of Molecular Biology, Lewis Thomas Lab, Princeton University, Princeton University, Infections Virales et Pathologie Comparée - UMR 754 (IVPC), Institut National de la Recherche Agronomique (INRA)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, L'Institut hospitalo-universitaire de Strasbourg (IHU Strasbourg), Institut National de Recherche en Informatique et en Automatique (Inria)-l'Institut de Recherche contre les Cancers de l'Appareil Digestif (IRCAD)-Les Hôpitaux Universitaires de Strasbourg (HUS)-La Fédération des Crédits Mutuels Centre Est (FCMCE)-L'Association pour la Recherche contre le Cancer (ARC)-La société Karl STORZ, Département d'écologie et évolution [Lausanne] (DEE), Université de Lausanne = University of Lausanne (UNIL), Biologie Computationnelle et Quantitative = Laboratory of Computational and Quantitative Biology (LCQB), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-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), Mathematical institute, Universiteit Leiden, Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), CAS Key Laboratory of Molecular Virology and Immunology [Shangai], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), French Agence Nationale de la Recherche sur le Sida et les hepatites virales [ANRS CSS4 AO2014-2q], FINOVI foundation, CAS 100 Talents, Shanghai municipality 1000 Talents, European Research Council [ERC-2008-AdG-233130-HEPCENT], National Institutes of Health [R01 AI079031], Research Scholar Award from the American Cancer Society [RSG-15-048-01-MPC], Burroughs Wellcome Fund Award for Investigators in Pathogenesis, French Ministry of Research (MESR), New Jersey Commission on Cancer Research [DHFS16PPC007], MAPPING project [ANR-11-BINF-0003], Institut Universitaire de France, Interreg IV-Hepato-Regio-Net, European Project: 305600,EC:FP7:HEALTH,FP7-HEALTH-2012-INNOVATION-1,HEPAMAB(2013), Carbone, Alessandra, Cosset, François-Loïc, Lavillette, Dimitri, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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 National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Lyon (ENVL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Institut National de la Recherche Agronomique (INRA)-École pratique des hautes études (EPHE)-Université Claude Bernard Lyon 1 (UCBL), Université de Lausanne (UNIL), Universiteit Leiden [Leiden], 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)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Ecole Nationale Vétérinaire de Lyon (ENVL), Institut National de la Recherche Agronomique (INRA)-École pratique des hautes études (EPHE), Sorbonne Université (SU)-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), Cheval, Christelle, and Human monoclonal antibody therapy to prevent hepatitis C virus reinfection of liver transplants: advancing lead monoclonal antibodies into clinical trial - HEPAMAB - - EC:FP7:HEALTH2013-01-01 - 2017-12-31 - 305600 - VALID
Subjects: 0301 basic medicine, Cell Membranes, Aucun, Hepacivirus, medicine.disease_cause, Inbred C57BL, Virus Replication, Membrane Fusion, Cell Fusion, Database and Informatics Methods, test in vitro, Mice, Spectrum Analysis Techniques, Viral Envelope Proteins, Tumor Cells, Cultured, Animals, Carcinoma, Hepatocellular/metabolism, Carcinoma, Hepatocellular/pathology, Carcinoma, Hepatocellular/virology, Evolution, Molecular, Hepacivirus/physiology, Hepatitis C/metabolism, Hepatitis C/pathology, Hepatitis C/virology, Humans, Liver Neoplasms/metabolism, Liver Neoplasms/pathology, Liver Neoplasms/virology, Mice, Inbred C57BL, Protein Binding, Viral Envelope Proteins/chemistry, Viral Envelope Proteins/genetics, Viral Envelope Proteins/metabolism, Virus Internalization, Enzyme-Linked Immunoassays, lcsh:QH301-705.5, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Cell fusion, Cultured, Microbiology and Parasitology, Liver Neoplasms, Flow Cytometry, Hepatitis C, Microbiologie et Parasitologie, hépatite c, 3. Good health, Tumor Cells, acide aminé, Spectrophotometry, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Cytophotometry, Cellular Structures and Organelles, Sequence Analysis, Research Article, lcsh:Immunologic diseases. Allergy, Cell Physiology, Evolutionary Processes, Carcinoma, Hepatocellular, [SDV.IMM] Life Sciences [q-bio]/Immunology, Bioinformatics, Viral protein, Evolution, Immunology, Computational biology, virus, Viral Structure, Sciences du Vivant [q-bio]/Médecine humaine et pathologie, Biology, Research and Analysis Methods, Microbiology, 03 medical and health sciences, Viral envelope, Viral entry, Virology, Genetics, medicine, Immunoassays, Molecular Biology, Coevolution, Evolutionary Biology, protéine glycosylée, Carcinoma, Biology and Life Sciences, Lipid bilayer fusion, Molecular, Hepatocellular, Cell Biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Viral replication, coévolution, lcsh:Biology (General), Immunologic Techniques, Parasitology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, lcsh:RC581-607, Sequence Alignment, Fusion mechanism
Abstract: Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses., Author summary Several virus-mediated molecular processes remain poorly described, which dampen the development of potent anti-viral therapies. Hence, new experimental strategies need to be undertaken to improve and accelerate our understanding of these processes. Here, as a proof of concept, we employ amino-acid coevolution as a tool to gain insights into the structural rearrangements of Hepatitis C Virus (HCV) envelope glycoproteins E1 and E2 during virus fusion with the cell membrane, and provide a basis for the inhibition of this process. Our coevolution analysis predicted that a specific domain of E2, the Back Layer (BL) is involved into significant conformational changes with E1 during the fusion of the HCV membrane with the cellular membrane. Consistently, a recombinant, soluble form of the BL was able to inhibit E1E2 fusogenic rearrangements and HCV infection. Moreover, predicted coevolution networks involving E1 and BL residues, as well as E1 and BL-adjacent residues, were found to modulate virus fusion. Our data shows that coevolution analysis is a powerful and underused approach that can provide significant insights into the functions and structural rearrangements of viral proteins. Importantly, this approach can also provide structural and molecular basis for the design of effective anti-viral drugs, and opens new perspectives to rapidly identify effective antiviral strategies against emerging and re-emerging viral pathogens.
Published: 2018

86. Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes

Author: Lucifora, Julie, Bonnin, Marc, Aillot, Ludovic, Fusil, Floriane, Maadadi, Sarah, Dimier, Laura, Michelet, Maud, Floriot, Océane, Ollivier, Anaïs, Rivoire, Michel, Ait-Goughoulte, Malika, Daffis, Stéphane, Fletcher, Simon P., Salvetti, Anna, François-Loïc, Cosset, Zoulim, Fabien, Durantel, David, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Application des ultrasons à la thérapie (LabTAU), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Roche Pharma Research and Early Development [Basel] (pRED), F. Hoffmann-La Roche [Basel], Gilead Sciences, Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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 Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Hepatitis B virus, lcsh:R, Toll-Like Receptors, NF-kappa B, lcsh:Medicine, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Ligands, Virus Replication, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Antiviral Agents, Article, Lipopeptides, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hepatocytes, Leukocytes, Mononuclear, [SDV.IMM]Life Sciences [q-bio]/Immunology, Humans, lcsh:Q, Interferons, lcsh:Science
Abstract: International audience; Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or "adjuvant" potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-\kappaB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.
Published: 2018

87. Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion

Author: Claire Montpellier, Gilles Duverlie, Marc le Maire, Cédric Montigny, Yann Ciczora, François-Loïc Cosset, Jean Dubuisson, Birke Andrea Tews, Pierre Falson, Laura Riva, Eve-Isabelle Pécheur, Antoine Loquet, Birke Bartosch, Khaled Alsaleh, François Penin, Bases moléculaires et structurales des systèmes infectieux (BMSSI), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), 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), Laboratoire des Protéines et Systèmes Membranaires (LPSM), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Unité de Virologie clinique et fondamentale (UVCF), Université de Picardie Jules Verne (UPJV)-CHU Amiens-Picardie, Virology Research Unit EA4294, Université de Picardie Jules Verne (UPJV), ANR-11-BINF-0003,MAPPING,Vers une cartographie haute résolution des interactions protéiques à l'échelle du génome.(2011), 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 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), Institut de biologie et chimie des protéines [Lyon] ( IBCP ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Centre International de Recherche en Infectiologie ( 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 ), Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 ( CIIL ), Réseau International des Instituts Pasteur ( RIIP ) -Réseau International des Instituts Pasteur ( RIIP ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -IFR142-Université de Lille-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire des Protéines et Systèmes Membranaires ( LPSM ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Unité de Virologie clinique et fondamentale EA 4294, CHU Amiens-Picardie, Jules Verne University of Picardie, Bases moléculaires et structurales des systèmes infectieux ( BMSSI ), and ANR-11-BINF-0003/11-BINF-0003,MAPPING,MAPPING ( 2011 )
Subjects: Models, Molecular, Recombinant Fusion Proteins, [SDV]Life Sciences [q-bio], Amino Acid Motifs, Molecular Sequence Data, Immunology, Gene Expression, Hepacivirus, Plasma protein binding, Biology, Microbiology, Protein structure, Viral Envelope Proteins, Viral entry, Virology, Escherichia coli, chemistry.chemical_classification, Binding Sites, [ SDV ] Life Sciences [q-bio], Protein Stability, Virus Assembly, Structure and Assembly, Virion, Virus Internalization, Fusion protein, Molecular biology, Transmembrane protein, Protein Structure, Tertiary, 3. Good health, Cell biology, Transmembrane domain, chemistry, Virion assembly, Insect Science, Mutation, Protein Multimerization, Glycoprotein, Sequence Alignment, Protein Binding
Abstract: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.
Published: 2015

88. Virology and cell biology of the hepatitis C virus life cycle – An update

Author: Jean Dubuisson, François-Loïc Cosset, 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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), LabEx ECOFECT Inserm U1111, Work in the JD laboratory was supported by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) and ANR through ERA-NET Infect-ERA program (ANR-13-IFEC-0002-01). Work in the FLC laboratory was supported by ANRS, the European Research Council (ERC-2008-AdG-233130-HEPCENT) and the LabEx ECOFECT (ANR-11-LABX-0048) of Université de Lyon., We thank Laurence Cocquerel and Marlène Dreux for critical reading of the manuscript and Sophana Ung for help with the Figures. We apologize to all colleagues whose work could not be referenced because of length restriction., 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 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cirrhosis, MESH: Virus Replication/physiology, [SDV]Life Sciences [q-bio], Hepacivirus, medicine.disease_cause, Virus Replication, MESH: Hepacivirus/growth & development, Virus entry, MESH: RNA, Viral/physiology, MESH: Hepatitis C/pathology, MESH: Cell Biology/trends, education.field_of_study, MESH: Life Cycle Stages/physiology, Hepatitis C virus, Viral life cycle, MESH: Hepacivirus/pathogenicity, virus diseases, MESH: Hepatitis C/physiopathology, Hepatitis C, 3. Good health, Interaction with host, Hepatocellular carcinoma, MESH: Hepacivirus/genetics, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, RNA, Viral, MESH: Virology/trends, Population, Biology, Virus-host interactions, MESH: Lipid Metabolism/physiology, Virology, medicine, Humans, education, Hepatitis, Life Cycle Stages, MESH: Humans, Hepatology, Cell Biology, medicine.disease, Lipid Metabolism, digestive system diseases, MESH: Hepatocytes/pathology, MESH: Hepatocytes/virology, Immunology, Hepatocytes, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Affiliations ECOFECT; International audience; Hepatitis C virus (HCV) is an important human pathogen that causes hepatitis, liver cirrhosis and hepatocellular carcinoma. It imposes a serious problem to public health in the world as the population of chronically infected HCV patients who are at risk of progressive liver disease is projected to increase significantly in the next decades. However, the arrival of new antiviral molecules is progressively changing the landscape of hepatitis C treatment. The search for new anti-HCV therapies has also been a driving force to better understand how HCV interacts with its host, and major progresses have been made on the various steps of the HCV life cycle. Here, we review the most recent advances in the fast growing knowledge on HCV life cycle and interaction with host factors and pathways.
Published: 2014
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89. Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

Author: Lévy, Camille, Amirache, Fouzia, Girard-Gagnepain, Anais, Frecha, Cecilia, Roman-Rodriguez, Francisco J., Bernadin, Ornellie, Costa, Caroline, Nègre, Didier, Gutierrez-Guerrero, Alejandra, Vranckx, Lenard S., Clerc, Isabelle, Taylor, Naomi, Thielecke, Lars, Cornils, Kerstin, Bueren, Juan A., Rio, Paula, Gijsbers, Rik, François-Loïc, Cosset, Verhoeyen, Els, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), LabEx ECOFECT Inserm U1111, Institute of Basic Sciences and Experimental Medicine, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas [Madrid] (CIEMAT), Instituto de Investigación Sanitaria Fundación Jiménez Diaz [Madrid] (IIS-FJD), Universidad Autonoma de Madrid (UAM)-Fundacion Jimenez Diaz [Madrid] (FJD), Centro de Investigación Biomédica En Red de Enfermedades Raras (CIBER-ER), Laboratories of Molecular Virology and Gene Therapy, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Laboratory of Excellence GR-Ex ' The red cell : from genesis to death ', PRES Sorbonne Paris Cité, Technische Universität Dresden = Dresden University of Technology (TU Dresden), Research Institute Children's Cancer Center, Hamburg, Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), Universidad Autónoma de Madrid (UAM)-Fundacion Jimenez Diaz [Madrid] (FJD), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)
Subjects: LENTIVIRAL VECTORS, CIENCIAS MÉDICAS Y DE LA SALUD, integumentary system, chemical and pharmacologic phenomena, hemic and immune systems, PSEUDOTYPE, Gene Therapy, bacterial infections and mycoses, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, complex mixtures, Biotecnología de la Salud, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, RAGA MICE, GENETHERAPY, Otras Biotecnologías de la Salud
Abstract: Hematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now. Fil: Lévy, Camille. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Amirache, Fouzia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Girard Gagnepain, Anais. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Universidad Escuela de Medicina del Hospital Italiano; Argentina Fil: Roman Rodríguez, Francisco J.. Consejo Superior de Investigaciones Científicas; España. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España. Instituto de Investigación Sanitaria Fundación Jiménez Díaz; España Fil: Bernadin, Ornellie. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Costa, Caroline. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Nègre, Didier. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Gutierrez Guerrero, Alejandra. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia Fil: Vranckx, Lenard S.. Katholikie Universiteit Leuven; Bélgica Fil: Clerc, Isabelle. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; Francia Fil: Taylor, Naomi. Université Montpellier II; Francia. Centre National de la Recherche Scientifique; Francia Fil: Thielecke, Lars. Technische Universität Dresden; Alemania Fil: Cornils, Kerstin. Research Institute Children’s Cancer Center Hamburg; Alemania Fil: Bueren, Juan A.. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España. Instituto de Investigación Sanitaria Fundación Jiménez Díaz; España. Consejo Superior de Investigaciones Científicas; España Fil: Rio, Paula. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España. Instituto de Investigación Sanitaria Fundación Jiménez Díaz; España. Consejo Superior de Investigaciones Científicas; España Fil: Gijsbers, Rik. Katholikie Universiteit Leuven; Bélgica Fil: Cosset, François Loïc. Centre National de la Recherche Scientifique; Francia. Université Claude Bernard Lyon 1; Francia. Inserm; Francia Fil: Verhoeyen, Els. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Inserm; Francia
Published: 2017

90. Efficient genome editing in primary cells and in vivo using viral-derived 'Nanoblades' loaded with Cas9/sgRNA ribonucleoproteins

Author: Emilie Laurent, Fabien Aube, Floriane Fusil, Théophile Ohlmann, Thibault J. M. Sohier, Emiliano P. Ricci, Aline Marnef, Emmanuelle Massouridès, Valérie Risson, Virginie Mournetas, Juliana Blin, Els Verhoeyen, Christian Pinset, François-Loïc Cosset, Antoine Corbin, Gaëlle Legube, Laurent Schaeffer, and Philippe E. Mangeot
Subjects: 0303 health sciences, Cas9, Biology, Molecular biology, Genome engineering, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, Haematopoiesis, 0302 clinical medicine, Genome editing, chemistry, In vivo, 030220 oncology & carcinogenesis, Stem cell, Gene, DNA, 030304 developmental biology
Abstract: Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.
Published: 2017

91. Baboon envelope pseudotyped lentiviral vectors: a highly efficient new tool to genetically manipulate T-cell acute lymphoblastic leukaemia-initiating cells

Author: Els Verhoeyen, Guillaume Hypolite, Vahid Asnafi, Ornellie Bernadin, Elisabeth Macintyre, Caroline Costa, Melania Tesio, Camille Lévy, François-Loïc Cosset, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cancer Research, T-Lymphocytes, Genetic Vectors, Cell Line, Fusion gene, 03 medical and health sciences, Transduction (genetics), Mice, Transduction, Genetic, Viral Envelope Proteins, Transduction, Genetic, Cell Line, Tumor, hemic and lymphatic diseases, biology.animal, medicine, Animals, Humans, ComputingMilieux_MISCELLANEOUS, Tumor, biology, Lentivirus, Gene Transfer Techniques, hemic and immune systems, Hematology, Genetic Therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma, medicine.disease, biology.organism_classification, Virology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Haematopoiesis, Leukemia, 030104 developmental biology, Oncology, Cell culture, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Stem cell, Baboon
Abstract: Baboon envelope pseudotyped lentiviral vectors: a highly efficient new tool to genetically manipulate T-cell acute lymphoblastic leukaemia-initiating cells
Published: 2017

92. A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

Author: Yu Yan, Xuesong Wang, Bertrand Boson, Jin Zhong, Ying He, François-Loïc Cosset, CAS Key Laboratory of Molecular Virology and Immunology [Shangai], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), 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), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, hepatitis C virus, Threonine, helix α0, NS3, viruses, Hepacivirus, Viral Nonstructural Proteins, medicine.disease_cause, Endoplasmic Reticulum, 0302 clinical medicine, Methionine, Virus Release, Mutation, biology, Viral Core Proteins, virus diseases, core, 3. Good health, Cell biology, Virion assembly, HCV, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030211 gastroenterology & hepatology, Viral genome replication, assembly, Immunology, Mutation, Missense, Microbiology, Cell Line, 03 medical and health sciences, Viral envelope, Virology, medicine, Humans, Point Mutation, Point mutation, Structure and Assembly, Virus Assembly, Helicase, biochemical phenomena, metabolism, and nutrition, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, digestive system diseases, 030104 developmental biology, Insect Science, biology.protein, Hepatocytes, Mutant Proteins, Missense
Abstract: The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.
Published: 2017

93. Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of Viral Genome to Assembly Sites

Author: Christophe Chamot, Solène Denolly, François-Loïc Cosset, Fanny Turlure, Marlène Dreux, Bertrand Boson, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), SFR Biosciences, 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), ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), European Project: 233130,EC:FP7:ERC,ERC-2008-AdG,HEPCENT(2009), 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), Boson, Bertrand, Dynamiques eco-évolutives des maladies infectieuses - - ECOFECT2011 - ANR-11-LABX-0048 - LABX - VALID, Molecular Analysis of Hepatitis C Virus Neutralization and Entry For the Development of Novel Antiviral Immunopreventive Strategies - HEPCENT - - EC:FP7:ERC2009-04-01 - 2014-03-31 - 233130 - VALID, and 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)
Subjects: 0301 basic medicine, Daclatasvir, Pyrrolidines, Hepatitis C virus, viruses, 030106 microbiology, Mutant, Genome, Viral, Hepacivirus, Biology, Viral Nonstructural Proteins, medicine.disease_cause, Antiviral Agents, RNA Transport, 03 medical and health sciences, Multiplicity of infection, Viral Envelope Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Cell Line, Tumor, medicine, Humans, chronic hepatitis C, NS5A, DAA, Infectivity, direct-acting antiviral agent, Hepatology, Endoplasmic reticulum, Viral Core Proteins, Virus Assembly, Gastroenterology, Imidazoles, virus diseases, Valine, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Virology, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, digestive system diseases, 3. Good health, Protein Transport, 030104 developmental biology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, RNA, Viral, Carbamates, Biogenesis, medicine.drug
Abstract: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs and inhibition of viral particle assembly., HCVcc particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity as well as production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy., Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes., In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins, because viral particles and replication complex vesicles cannot form and/or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.
Published: 2016

94. Generation of transgenic mice expressing EGFP protein fused to NP68 MHC class I epitope using lentivirus vectors

Author: Denise Aubert, Martine Tomkowiak, Jean-Louis Thoumas, Charles-Antoine Coupet, Didier Nègre, Marie Teixeira, Molly Brunner, Raffaella Ghittoni, François-Loïc Cosset, Jacqueline Marvel, Yann Leverrier, Judit Szécsi, Bariza Blanquier, and Els Verhoeyen
Subjects: 0303 health sciences, biology, T cell, Antigen presentation, Cross-presentation, Priming (immunology), Cell Biology, Molecular biology, Epitope, Immune tolerance, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, medicine.anatomical_structure, MHC class I, Genetics, biology.protein, medicine, Cytotoxic T cell, 030304 developmental biology, 030215 immunology
Abstract: Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken β-actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8α+ cDCs, and CD8α− cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here arevaluable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs. genesis 51:193–200, 2013. © 2012 Wiley Periodicals, Inc.
Published: 2013

95. Tumor antigen–specific induction of transcriptionally targeted retroviral vectors from chimeric immune receptor–modified T cells

Author: John, Chester, Anja, Ruchatz, Michael, Gough, Marka, Crittenden, Heung, Chong, François-Loïc, Cosset, François, Loïc-Cosset, Rosa Maria, Diaz, Kevin, Harrington, Luis, Alvarez-Vallina, and Richard, Vile
Subjects: Time Factors, Transcription, Genetic, T-Lymphocytes, Genetic enhancement, Genetic Vectors, Green Fluorescent Proteins, Biomedical Engineering, Mice, Nude, Bioengineering, Immune receptor, Biology, Polymerase Chain Reaction, Applied Microbiology and Biotechnology, Jurkat cells, Adenoviridae, Viral vector, Jurkat Cells, Mice, Immune system, Cell Adhesion, Tumor Cells, Cultured, Animals, Humans, Vector (molecular biology), Models, Genetic, T-cell receptor, Genetic Therapy, Flow Cytometry, Virology, Tumor antigen, Protein Structure, Tertiary, Luminescent Proteins, Retroviridae, Cancer research, Molecular Medicine, Biotechnology
Abstract: High-level systemic delivery of viral vectors to tumors has proved problematic as a result of immune neutralization, nonspecific adhesion, and clearance of circulating viral particles. Some cell types localize to tumors in response to particular biological properties associated with tumor growth. Their use to deliver viral vectors to tumors would allow precious viral stocks to be protected until they can be released at high local concentrations. Here, we describe a mechanism by which retroviral vector production by T cells can be regulated by a tumor-specific trigger through engagement of a chimeric immune receptor (CIR) with its target antigen. The virus that is released from the T cells can also be transcriptionally targeted. Finally, we show that it is possible to use vector-loaded, antigen-triggered human T cells as therapeutic, tumor-specific vector delivery cells in models of both local intratumoral and systemic delivery to both lung and liver metastases. This strategy incorporates multiple levels of targeting into the delivery system at the stages of surface targeting, viral production, and gene expression.
Published: 2002

96. Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication

Author: David Durantel, Fouzia Amirache, François-Loïc Cosset, Adrien Foca, Thomas Lahlali, Pascal Jalaguier, Nathalie Isorce, Ahmed Diab, Fabien Zoulim, Lia N'Guyen, Floriane Fusil, Ourania M. Andrisani, Service d'Hépatologie [Hôpital de la Croix-Rousse - HCL], Hôpital de la Croix-Rousse [CHU - HCL], Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0301 basic medicine, Risk, Small interfering RNA, Hepatitis B virus, Carcinogenesis, Cells, Primary Cell Culture, Drug Evaluation, Preclinical, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cell Cycle Proteins, Biology, Protein Serine-Threonine Kinases, medicine.disease_cause, Virus Replication, Hepatitis B virus PRE beta, Virus, Hepatitis, 03 medical and health sciences, Mice, In vivo, Proto-Oncogene Proteins, medicine, Animals, Humans, Amino Acid Sequence, Phosphorylation, Host factor, Hepatology, Kinase, Pteridines, Viral Core Proteins, Carcinoma, virus diseases, Dna, Hepatitis B, Virology, digestive system diseases, 3. Good health, Enzyme Activation, 030104 developmental biology, Viral replication, Liver, Mutagenesis, Host-Pathogen Interactions, Hepatocytes, France, biosynthesis, Infection
Abstract: Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA ). In addition, a humanized liver Fah-/- /Rag2-/- /Il2rg-/- (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. Conclusion PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).
Published: 2016

97. Triggering the TCR Developmental Checkpoint Activates a Therapeutically Targetable Tumor Suppressive Pathway in T-cell Leukemia

Author: David-Alexandre Gross, Melania Tesio, Amélie Trinquand, Mohamed Belhocine, Hervé Dombret, Norbert Ifrah, Benedetta Zaniboni, Lucienne Chatenoud, Vahid Asnafi, Francesca Rocchetti, François-Loïc Cosset, Nuno R. dos Santos, Christine Tran Quang, Ludovic Lhermitte, Jacques Ghysdael, Elizabeth Macintyre, Michael Dussiot, Françoise Pflumio, Olivier Hermine, Els Verhoeyen, Cindy Da Costa de Jesus, Salvatore Spicuglia, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Algarve [Portugal], Stress génotoxiques et cancer, Université Paris-Sud - Paris 11 (UP11)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Technologies avancées pour le génôme et la clinique (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratory of molecular mechanisms of hematologic disorders and therapeutic implications (ERL 8254 - Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Equipe 'Contrôle Métabolique des Morts Cellulaires' (INSERM U1065 - C3M), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Laboratoire de Recherche sur les Cellules Souches Hématopoiétiques et Leucémiques (LSHL), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7), Tolérance immunitaire et présentation antigénique: impact en auto-immunité et en transplantation, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service d'immuno-hématologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Necker Enfants-Malades (INEM) ( INEM - UM 111 (UMR 8253 / U1151) ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Technologies avancées pour le génôme et la clinique ( TAGC ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Excellence GR-Ex, Sorbonne Paris Cité-Université Paris Descartes - Paris 5 ( UPD5 ) -Imagine Institute, Centre International de Recherche en Infectiologie ( 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 ), Boehringer Ingelheim RCV, Service de Chimie Moléculaire ( SCM ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ), Hematology Department, Université d'Angers ( UA ), Centre de Recherche en Cancérologie / Nantes - Angers ( CRCNA ), CHU Angers-Centre hospitalier universitaire de Nantes ( CHU Nantes ) -Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Hôpital Laennec-Centre National de la Recherche Scientifique ( CNRS ) -Faculté de Médecine d'Angers, Hôpital Saint-Louis, Assistance publique - Hôpitaux de Paris (AP-HP)-Université Paris Diderot - Paris 7 ( UPD7 ), Imagine - Institut des maladies génétiques ( IMAGINE - U1163 ), Service d'hématologie-immunologie-oncologie pédiatrique, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Université Paris-Sud - Paris 11 (UP11)-Institut Curie-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), 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), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie / Nantes - Angers (CRCNA), Centre hospitalier universitaire de Nantes (CHU Nantes)-Faculté de Médecine d'Angers-Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Centre National de la Recherche Scientifique (CNRS)-Hôpital Laennec-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôtel-Dieu de Nantes, Service d'hématologie-immunologie-oncologie pédiatrique [CHU Trousseau], Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Necker - Enfants Malades [AP-HP], Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Paris-Sud - Paris 11 (UP11), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Trousseau [APHP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)
Subjects: 0301 basic medicine, Leukemia, T-Cell, CD3 Complex, medicine.medical_treatment, CD3, T-Lymphocytes, [SDV]Life Sciences [q-bio], T-cell leukemia, Receptors, Antigen, T-Cell, Antigen-Presenting Cells, Apoptosis, Biology, Lymphocyte Activation, Targeted therapy, Immunophenotyping, 03 medical and health sciences, Mice, medicine, Animals, Humans, Clonal Selection, Antigen-Mediated, Mice, Knockout, [ SDV ] Life Sciences [q-bio], T-cell receptor, Antibodies, Monoclonal, medicine.disease, 3. Good health, Leukemia, Disease Models, Animal, 030104 developmental biology, Oncology, Tumor progression, Immunology, Cancer cell, biology.protein, Female, Signal Transduction
Abstract: Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ϵ chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. Significance: T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972–85. ©2016 AACR. See related commentary by Lemonnier and Mak, p. 946. This article is highlighted in the In This Issue feature, p. 932
Published: 2016

98. Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells

Author: Morozumi, Yuichi, Boussouar, Fayçal, Tan, Minjia, Chaikuad, Apirat, Jamshidikia, Mahya, Colak, Gozde, He, Huang, Nie, Litong, Petosa, Carlo, De Dieuleveult, Maud, Curtet, Sandrine, Vitte, Anne-Laure, Rabatel, Clothilde, Debernardi, Alexandra, François-Loïc, Cosset, Verhoeyen, Els, Emadali, Anouk, Schweifer, Norbert, Gianni, Davide, Gut, Marta, Guardiola, Philippe, Rousseaux, Sophie, Gérard, Matthieu, Knapp, Stefan, Zhao, Yingming, Khochbin, Saadi, Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), The Chemical Proteomics Center and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Structural Genomics Consortium, University of Oxford, Nuffield Department of Clinical Medicine [Oxford], Ben May Department of Cancer Research, The University of Chicago Medicine, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), 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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Equipe 'Contrôle Métabolique des Morts Cellulaires' (INSERM U1065 - C3M), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Boehringer Ingelheim RCV, Centre for Genomic Regulation [Barcelona] (CRG), Universitat Pompeu Fabra [Barcelona] (UPF)-Centro Nacional de Analisis Genomico [Barcelona] (CNAG), Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM)-PRES Université Nantes Angers Le Mans (UNAM)-Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hôpital Laennec-Centre National de la Recherche Scientifique (CNRS)-Faculté de Médecine d'Angers-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut d'oncologie/développement Albert Bonniot de Grenoble ( INSERM U823 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale ( INSERM ), University of Oxford [Oxford], Target Discovery Institute (TDI), Nuffield Department of Clinical Medicine, Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Centre International de Recherche en Infectiologie ( CIRI ), Centre National de la Recherche Scientifique ( CNRS ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-École normale supérieure - Lyon ( ENS Lyon ), Centre for Genomic Regulation [Barcelona] ( CRG ), Universitat Pompeu Fabra [Barcelona]-Centro Nacional de Analisis Genomico [Barcelona] ( CNAG ), Inserm u892, Centre de Recherches en Cancérologie, Nantes, Institut National de la Santé et de la Recherche Médicale (INSERM)-EFS-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), 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), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Adenosine Triphosphatases, Male, Proteomics, histone turnover, Chromatin Immunoprecipitation, germ cells, Genome, Pax3, [ SDV ] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], FACT, epidrug, Acetylation, Cell Differentiation, Article, Chromatin, Nucleosomes, DNA-Binding Proteins, histone chaperone, ATPases Associated with Diverse Cellular Activities, Humans, cancer drug target, Embryonic Stem Cells, Cell Proliferation, Protein Binding
Abstract: International audience; Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.
Published: 2016

99. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms

Author: Florence Nguyen-Khac, Sophie Park, Isabelle Templier, Martine Chauvet, Joel Plumas, Julie Charles, Dominique Leroux, Christine Lefebvre, Sophie Rousseaux, Sieme Hamaidia, François-Loïc Cosset, Els Verhoeyen, Mary Callanan, Dominique Penther, Marie-Christine Jacob, Thierry Bonnefoix, Isabelle Luquet, Sarah Bertrand, Anne Roggy, Cornelis P. Tensen, Elizabeth Macintyre, Anouk Emadali, Neda Hoghoughi, Elisabeth Brambilla, Remy Gressin, Laurence Chaperot, Azadeh Hajmirza, Takahiro Maeda, Christophe Roumier, Philippe Bertrand, Céline Suchaud-Martin, Francine Garnache, Christian Bastard, Samuel Duley, Cristina Mecucci, Fabrice Jardin, Juliana Bruder-Costa, Véronique Josserand, 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)-Centre National de la Recherche Scientifique (CNRS)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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 méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Groupe d'étude des proliférations lymphoïdes (GPL), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'Hématologie Cellulaire [Lille], Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Etablissement français du sang [Bourgogne-Franche-Comté] (EFS [Bourgogne-Franche-Comté]), CHU de Grenoble-Alpes, Graduate School of Biomedical Sciences [Nagasaki University, Japan], Nagasaki University, Etablissement français du sang - Auvergne-Rhône-Alpes (EFS), Leiden University Medical Center (LUMC), Perugia University School of Medicine, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Laboratoire d'Hématologie [Purpan], Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse], 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 [2016-2019] (UGA [2016-2019]), 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), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Etablissement français du sang [Bourgogne-Franche-Comté] (EFS BFC), Universiteit Leiden, Università degli Studi di Perugia = University of Perugia (UNIPG), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service Hématologie - IUCT-Oncopole [CHU Toulouse], Pôle Biologie [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Pôle IUCT [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), 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), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Etablissement français du sang [Bourgogne-France-Comté] (EFS [Bourgogne-France-Comté]), and Groupe hospitalier Pitié- Salpêtrière, AP-HP
Subjects: 0301 basic medicine, Adult, Skin Neoplasms, Adolescent, Immunology, Plasmacytoid dendritic cell, Haploinsufficiency, Biology, Biochemistry, 03 medical and health sciences, Young Adult, Receptors, Glucocorticoid, medicine, Tumor Cells, Cultured, Humans, Neoplasm Invasiveness, Child, Gene, Aged, Genetics, Regulation of gene expression, Aged, 80 and over, Leukemia, Myeloid Neoplasia, Gene Expression Regulation, Leukemic, Point mutation, hemic and immune systems, Hematology, Cell Biology, Dendritic Cells, Middle Aged, medicine.disease, Non-coding RNA, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Gene expression profiling, stomatognathic diseases, 030104 developmental biology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Cancer research, [SDV.IMM]Life Sciences [q-bio]/Immunology, RNA, Long Noncoding
Abstract: International audience; Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressiveleukemia for which knowledge on disease mechanisms and effective therapies are currentlylacking. Only a handful of recurring genetic mutations have been identified and none isspecific to BPDCN. In this study, through molecular cloning in an index case that presenteda balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, weidentify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR),in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiencyfor NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely pooroverall survival (P 5 .0006). Consistent with a role for GCR in tumor suppression, functionalanalyses coupled with gene expression profiling identified corticoresistance and loss-ofEZH2 function as major downstream consequences of NR3C1 deletion in BPDCN.Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to along noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncodingRNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F.Overexpression oflincRNA-3qwas a consistent feature ofmalignant cells and could be abrogated by bromodomain and extraterminal domain(BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN andidentifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN. (
Published: 2016

100. Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD/SCIDγc-/- mice

Author: Nisha Nair, Thierry VandenDriessche, Didier Nègre, François-Loïc Cosset, Girard-Gagnepain A, Camille Lévy, Fouzia Amirache, Antonio Rodríguez, Els Verhoeyen, Guillermo Garaulet, Marinee Khim Chuah, Ornellie Bernadin, Floriane Fusil, Caroline Costa, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), 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), Universidad Autonoma de Madrid (UAM), Université libre de Bruxelles (ULB), Center for Molecular and Vascular Biology, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Informatics and Applied Informatics, Basic (bio-) Medical Sciences, Division of Gene Therapy & Regenerative Medicine, 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), Universidad Autónoma de Madrid (UAM), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)
Subjects: 0301 basic medicine, CD4-Positive T-Lymphocytes, Adoptive cell transfer, Genetic enhancement, Mice, SCID, Factor IX, Mice, Viral Envelope Proteins, Mice, Inbred NOD, Transduction, Genetic, hemophilia, Transgenes, Clotting factor, Medicine(all), B-Lymphocytes, Membrane Glycoproteins, Gene Transfer Techniques, Hematology, gene therapy, Cell biology, medicine.anatomical_structure, Treatment Outcome, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, NSG mouse, [SDV.IMM]Life Sciences [q-bio]/Immunology, Immunotherapy, Plasmids, Genetic Vectors, Antigen-Presenting Cells, Biology, SCID, Hemophilia A, Viral vector, 03 medical and health sciences, Transduction, Genetic, medicine, Animals, b cells, Humans, Antigen-presenting cell, B cell, Cell Proliferation, Lentivirus, lentiviral vector, Virology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, HEK293 Cells, Inbred NOD, Ex vivo, Papio
Abstract: International audience; Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. SUMMARY: Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc-/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc-/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.
Published: 2016

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