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1. Spinal cord injury dysregulates fibro-adipogenic progenitors miRNAs signaling to promote neurogenic heterotopic ossifications

Author

Jules Gueguen, Dorothée Girard, Bastien Rival, Juliette Fernandez, Marie-Emmanuelle Goriot, and Sébastien Banzet

Subjects
Biology (General), QH301-705.5
Abstract

Abstract Neurogenic heterotopic ossifications are intramuscular bone formations developing following central nervous system injury. The pathophysiology is poorly understood and current treatments for this debilitating condition remain unsatisfying. Here we explored the role of miRNAs in a clinically relevant mouse model that combines muscle and spinal cord injury, and in patients’ cells. We found an osteo-suppressive miRNAs response in injured muscle that was hindered when the spinal cord injury was associated. In isolated fibro-adipogenic progenitors from damaged muscle (cells at the origin of ossification), spinal cord injury induced a downregulation of osteo-suppressive miRNAs while osteogenic markers were overexpressed. The overexpression of selected miRNAs in patient’s fibro-adipogenic progenitors inhibited mineralization and osteo-chondrogenic markers in vitro. Altogether, we highlighted an osteo-suppressive mechanism involving multiple miRNAs in response to muscle injury that prevents osteogenic commitment which is ablated by the neurologic lesion in heterotopic ossification pathogenesis. This provides new research hypotheses for preventive treatments.

Published
2023
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2. Endogenous TRIM5α Function Is Regulated by SUMOylation and Nuclear Sequestration for Efficient Innate Sensing in Dendritic Cells

Author

Débora M. Portilho, Juliette Fernandez, Mathieu Ringeard, Anthony K. Machado, Aude Boulay, Martha Mayer, Michaela Müller-Trutwin, Anne-Sophie Beignon, Frank Kirchhoff, Sébastien Nisole, and Nathalie J. Arhel

Subjects
Biology (General), QH301-705.5
Abstract

During retroviral infection, viral capsids are subject to restriction by the cellular factor TRIM5α. Here, we show that dendritic cells (DCs) derived from human and non-human primate species lack efficient TRIM5α-mediated retroviral restriction. In DCs, endogenous TRIM5α accumulates in nuclear bodies (NB) that partly co-localize with Cajal bodies in a SUMOylation-dependent manner. Nuclear sequestration of TRIM5α allowed potent induction of type I interferon (IFN) responses during infection, mediated by sensing of reverse transcribed DNA by cGAS. Overexpression of TRIM5α or treatment with the SUMOylation inhibitor ginkgolic acid (GA) resulted in enforced cytoplasmic TRIM5α expression and restored efficient viral restriction but abrogated type I IFN production following infection. Our results suggest that there is an evolutionary trade-off specific to DCs in which restriction is minimized to maximize sensing. TRIM5α regulation via SUMOylation-dependent nuclear sequestration adds to our understanding of how restriction factors are regulated.

Published
2016
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3. Daxx Inhibits HIV-1 Reverse Transcription and Uncoating in a SUMO-Dependent Manner

Author

Sarah Maillet, Juliette Fernandez, Mathilde Decourcelle, Khadija El Koulali, Fabien P. Blanchet, Nathalie J. Arhel, Ghizlane Maarifi, and Sébastien Nisole

Subjects
Daxx, HIV-1, reverse transcription, uncoating, restriction factor, interferon, Microbiology, QR1-502
Abstract

Death domain-associated protein 6 (Daxx) is a multifunctional, ubiquitously expressed and highly conserved chaperone protein involved in numerous cellular processes, including apoptosis, transcriptional repression, and carcinogenesis. In 2015, we identified Daxx as an antiretroviral factor that interfered with HIV-1 replication by inhibiting the reverse transcription step. In the present study, we sought to unravel the molecular mechanism of Daxx-mediated restriction and, in particular, to identify the protein(s) that Daxx targets in order to achieve its antiviral activity. First, we show that the SUMO-interacting motif (SIM) located at the C-terminus of the protein is strictly required for Daxx to inhibit HIV-1 reverse transcription. By performing a quantitative proteomic screen combined with classical biochemical analyses, we found that Daxx associated with incoming HIV-1 cores through a SIM-dependent interaction with cyclophilin A (CypA) and capsid (CA). Daxx was found to reside within a multiprotein complex associated with viral capsids, also containing TNPO3, TRIM5α, and TRIM34. Given the well-known influence of these cellular factors on the stability of HIV-1 cores, we investigated the effect of Daxx on the cytoplasmic fate of incoming cores and found that Daxx prevented HIV-1 uncoating in a SIM-dependent manner. Altogether, our findings suggest that, by recruiting TNPO3, TRIM5α, and TRIM34 and possibly other proteins onto incoming HIV-1 cores through a SIM-dependent interaction with CA-bound CypA, Daxx increases their stability, thus preventing uncoating and reverse transcription. Our study uncovers a previously unknown function of Daxx in the early steps of HIV-1 infection and further illustrates how reverse transcription and uncoating are two tightly interdependent processes.

Published
2020
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5. Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration.

Author

Francesca Di Nunzio, Anne Danckaert, Thomas Fricke, Patricio Perez, Juliette Fernandez, Emmanuelle Perret, Pascal Roux, Spencer Shorte, Pierre Charneau, Felipe Diaz-Griffero, and Nathalie J Arhel

Subjects
Medicine, Science
Abstract

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

Published
2012
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6. Identifying enhancers of innate immune signaling as broad-spectrum antivirals active against emerging viruses

Author

Ghizlane Maarifi, Marie-France Martin, Abderezak Zebboudj, Aude Boulay, Pierre Nouaux, Juliette Fernandez, Justine Lagisquet, Dominique Garcin, Raphael Gaudin, Nathalie J. Arhel, and Sébastien Nisole

Subjects
Pharmacology, SARS-CoV-2, Virus Diseases, Clinical Biochemistry, Drug Discovery, COVID-19, Humans, Molecular Medicine, Antiviral Agents, Molecular Biology, Biochemistry, Immunity, Innate
Abstract

The increasingly frequent outbreaks of pathogenic viruses have underlined the urgent need to improve our arsenal of antivirals that can be deployed for future pandemics. Innate immunity is a powerful first line of defense against pathogens, and compounds that boost the innate response have high potential to act as broad-spectrum antivirals. Here, we harnessed localization-dependent protein-complementation assays (called Alpha Centauri) to measure the nuclear translocation of interferon regulatory factors (IRFs), thus providing a readout of innate immune activation following viral infection that is applicable to high-throughput screening of immunomodulatory molecules. As proof of concept, we screened a library of kinase inhibitors on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identified Gilteritinib as a powerful enhancer of innate responses to viral infection. This immunostimulatory activity of Gilteritinib was found to be dependent on the AXL-IRF7 axis and results in a broad and potent antiviral activity against unrelated RNA viruses.

Published
2022

7. Measuring the subcellular compartmentalization of viral infections by protein complementation assay

Author

Sébastien Nisole, Virginie Georget, Cedric Hassen-Khodja, Yves Jacob, Yves L. Janin, Nathalie J. Arhel, Pierre-Olivier Vidalain, Thierry Rose, Juliette Fernandez, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM), BioCampus Montpellier (BCM), Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Université de Paris (UP), Chimie et Biocatalyse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Biologie cellulaire des infections virales – Cell biology of viral infection, 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), This work was financed by Sidaction, the Société d’Accélération de Transfert Technologique IdfInnov (now Erganeo), the Labex EpiGenMed 'Investissements d’avenir' ANR-10-LABX-12-01, the ATIP-Avenir program, and the Agence Nationale de Recherche sur le Sida et les Hépatites Virales., ANR-10-LABX-0012,EpiGenMed,From Genome and Epigenome to Molecular Medicine: turning new paradigms in biology into the therapeutic strategies of tomorrow(2010), Regional Imaging Platform, Montpellier Ressources Imagerie (MRI), BioCampus, Montpellier, France, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], 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), This work was financed by Sidaction, the Société d’Accélération de Transfert Technologique IdfInnov (now Erganeo), the Labex EpiGenMed 'Investissements d’avenir' ANR-10-LABX-12-01, the ATIP-Avenir program, and the Agence Nationale de Recherche sur le Sida et les Hépatites Virales. We thank Leslie Bancel-Vallee (Montpellier Ressources Imagerie, Montpellier) for help with the Airyscan imaging., BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-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), Arhel, Nathalie, and Laboratoires d'excellence - From Genome and Epigenome to Molecular Medicine: turning new paradigms in biology into the therapeutic strategies of tomorrow - - EpiGenMed2010 - ANR-10-LABX-0012 - LABX - VALID

Subjects
Cytoplasm, Nuclear Envelope, [SDV]Life Sciences [q-bio], Protein combining, Population, Green Fluorescent Proteins, HIV Infections, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Virus Replication, Green fluorescent protein, Cell Line, Small hairpin RNA, 03 medical and health sciences, Humans, viruses, Nuclear pore, education, 030304 developmental biology, Cell Nucleus, 0303 health sciences, education.field_of_study, Multidisciplinary, Nucleoplasm, biology, Chemistry, 030302 biochemistry & molecular biology, Biological Sciences, ubcellular compartments, Integrase, Cell biology, High-Throughput Screening Assays, [SDV] Life Sciences [q-bio], Viral replication, Ribonucleoproteins, Virus Diseases, technological development, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, Nuclear Pore, HeLa Cells
Abstract

International audience; The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (

Published
2021

8. [HIV capsid and productive infection: taking steps in the right direction]

Author

Juliette, Fernandez and Nathalie J, Arhel

Abstract

The Human Immunodeficiency Virus Type-1, the causative agent of AIDS, reaches its site of replication by trafficking through the cytoplasm towards the nucleus, benefiting from an active nuclear import through the nuclear pore in order to integrate in the genome of the host cell. Although it is generally accepted that the viral genome remains within the viral capsid for some time after cell entry, the mechanisms responsible for controlled uncoating, which is essential for productive infection, remain poorly understood. Numerous studies now show that the integrity of the viral capsid is essential for transport towards the nucleus, for reverse transcription and nuclear import, and to prevent sensing by innate immune receptors. This review aims to report recent developments in our understanding of the early stages of HIV infection, from entry into the cell to integration, highlighting the cellular partners of the HIV-1 capsid that promote or antagonize infection, as well as the different techniques that are developed for fundamental research and the identification of potential therapeutic targets.

Published
2020

9. Coordination de la décapsidation et de l’import nucléaire de VIH-1 par la transportine-1

Author

Juliette Fernandez, Nathalie J. Arhel, École Polytechnique de Montréal (EPM), Institut de Recherche en Infectiologie de Montpellier (IRIM), and Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, 03 medical and health sciences, 030306 microbiology, Chemistry, [SDV]Life Sciences [q-bio], General Medicine, Molecular biology, General Biochemistry, Genetics and Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

International audience; No abstract available

Published
2020

10. Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating

Author

Corinne Henriquet, Martine Pugnière, Camille Garcia, Thibaut Léger, Mickaël Blaise, Yohei Yamauchi, Juliette Fernandez, Débora M. Portilho, Sébastien Nisole, Laurent Chaloin, Anthony K. Machado, Célia Chamontin, Nathalie J. Arhel, Delphine Muriaux, Sébastien Lyonnais, Kathleen Gärtner, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Génétique et Ecologie des Virus, Génétique des Virus et Pathogénèse des Maladies Virales, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), Laboratoire d'Electronique Moléculaire (LEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Thérapie des maladies du muscle strié, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Toxicité environnementale, cibles thérapeutiques, signalisation cellulaire (T3S - UMR_S 1124), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Universitätsklinikum Ulm - University Hospital of Ulm, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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 National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Microbiology (medical), CD4-Positive T-Lymphocytes, Models, Molecular, Protein Conformation, viruses, [SDV]Life Sciences [q-bio], Immunology, Nuclear Localization Signals, Active Transport, Cell Nucleus, HIV Infections, Applied Microbiology and Biotechnology, Microbiology, Mass Spectrometry, 03 medical and health sciences, Protein structure, Capsid, Virus Uncoating, Genetics, medicine, Humans, Nuclear pore, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Binding Sites, 030306 microbiology, Chemistry, Cell Biology, beta Karyopherins, 3. Good health, Cell biology, Cell nucleus, medicine.anatomical_structure, HEK293 Cells, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Transportin 1, HIV-1, RNA, Viral, Capsid Proteins, Nuclear transport, Nuclear localization sequence, Gene Deletion, HeLa Cells, Protein Binding
Abstract

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify β-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.

Published
2019

11. [SUMO regulates the capacity of TRIM5α to inhibit HIV-1]

Author

Ghizlane Maarifi, Nathalie J. Arhel, Juliette Fernandez, Sébastien Nisole, Institut de Recherche en Infectiologie de Montpellier (IRIM), and Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, 03 medical and health sciences, Chemistry, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, [SDV.IMM]Life Sciences [q-bio]/Immunology, General Medicine, Molecular biology, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, General Biochemistry, Genetics and Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

International audience

Published
2019

12. Endogenous TRIM5α Function Is Regulated by SUMOylation and Nuclear Sequestration for Efficient Innate Sensing in Dendritic Cells

Author

Débora M. Portilho, Juliette Fernandez, Mathieu Ringeard, Anthony K. Machado, Aude Boulay, Martha Mayer, Michaela Müller-Trutwin, Anne-Sophie Beignon, Frank Kirchhoff, Sébastien Nisole, Nathalie J. Arhel, Génétique et Ecologie des Virus, Génétique des Virus et Pathogénèse des Maladies Virales, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), Virologie moléculaire et Vectorologie, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Universität Ulm - Ulm University [Ulm, Allemagne], HIV, Inflammation et persistance, Institut Pasteur [Paris], Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Toxicité environnementale, cibles thérapeutiques, signalisation cellulaire (T3S - UMR_S 1124), 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), This work was supported by grants from the Agence Nationale de Recherches sur le SIDA et les hépatites virales (ANRS), the ATIP-Avenir program (INSERM), the Deutsche Forschungsgemeinschaft (DFG), European FP7 'HIT HIDDEN HIV' (305762), a DFG Leibniz award, and an Advanced ERC Investigator grant (to F.K.)., For veterinary support, NHP welfare, and biological specimens sampling, we thank Nathalie Dereuddre-Bosquet and Céline Gommet (CEA Fontenay-aux-Roses) for RM and CM, Thierry Petit (Zoo de la Palmyre, Les Mathes) for PTM, and Anne-Sophie Liovat for AGM. We thank Paul Bieniasz (Aaron Diamond AIDS Research Center) for the rabbit TRIM5α antibody, Nicolas Manel (Institut Curie) for the cGAS shRNA construct, Cyril Pantoli for graphic design, and Ron Hay (University of Dundee) and Allan Hance (Hôpital Saint-Louis) for helpful discussions. Protein diagrams were prepared using Illustrator for Biological Sequences. Microscopy experiments were performed at the Imagopole (Institut Pasteur) and the technology platform of Hôpital Saint-Louis., European Project: 305762,HEALTH,FP7-HEALTH-2012-INNOVATION-1,HIT HIDDEN HIV(2012), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
MESH: Humans, MESH: Dendritic Cells, MESH: Sumoylation, [SDV]Life Sciences [q-bio], Ubiquitin-Protein Ligases, Proteins, Sumoylation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Dendritic Cells, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Macaca mulatta, Article, MESH: Macaca mulatta, lcsh:Biology (General), [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Animals, Humans, MESH: Animals, MESH: Proteins, lcsh:QH301-705.5
Abstract

Summary During retroviral infection, viral capsids are subject to restriction by the cellular factor TRIM5α. Here, we show that dendritic cells (DCs) derived from human and non-human primate species lack efficient TRIM5α-mediated retroviral restriction. In DCs, endogenous TRIM5α accumulates in nuclear bodies (NB) that partly co-localize with Cajal bodies in a SUMOylation-dependent manner. Nuclear sequestration of TRIM5α allowed potent induction of type I interferon (IFN) responses during infection, mediated by sensing of reverse transcribed DNA by cGAS. Overexpression of TRIM5α or treatment with the SUMOylation inhibitor ginkgolic acid (GA) resulted in enforced cytoplasmic TRIM5α expression and restored efficient viral restriction but abrogated type I IFN production following infection. Our results suggest that there is an evolutionary trade-off specific to DCs in which restriction is minimized to maximize sensing. TRIM5α regulation via SUMOylation-dependent nuclear sequestration adds to our understanding of how restriction factors are regulated., Graphical Abstract, Highlights • Primate dendritic cells (DCs) lack efficient TRIM5α-mediated retroviral restriction • In DCs TRIM5α is sequestered in the nucleus in a SUMOylation-dependent manner • TRIM5α nuclear sequestration allows DC sensing of retroviral DNA by cGAS, Retroviruses can be efficiently blocked by a cellular restriction factor called TRIM5α. Intriguingly, TRIM5α is inactive in dendritic cells (DCs). Portilho et al. show that in DCs TRIM5α is sequestered in the nucleus in a SUMOylation-dependent manner, favoring innate sensing of retroviruses in the cytoplasm by cGAS and thus an antiviral response.

Published
2015

14. Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

Author

Pierre Charneau, Anaba Zambo, Débora M. Portilho, Spencer L. Shorte, Juliette Fernandez, Sandie Munier, Pierre-Olivier Vidalain, Yves Jacob, Andreas Becker, François Clavel, Nathalie J. Arhel, Pascal Roux, Anne Danckaert, Génétique et Ecologie des Virus, Génétique des Virus et Pathogénèse des Maladies Virales, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagopole (CITECH), Institut Pasteur [Paris] (IP), 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), Génomique Virale et Vaccination, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Virologie moléculaire et vaccinologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), Institut Pasteur [Paris], Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]

Subjects
Microtubule-associated protein, [SDV]Life Sciences [q-bio], Active Transport, Cell Nucleus, HIV Core Protein p24, Transport, Microtubule, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Microfilament, Cell morphology, Microtubules, Biochemistry, Cell Line, 03 medical and health sciences, Virology, medicine, Humans, Nuclear membrane, Cytoskeleton, Molecular Biology, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Human Immunodeficiency Virus (HIV), 030306 microbiology, Macrophages, Microfilament Proteins, Cell Biology, 3. Good health, Cell biology, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Cytoplasm, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HIV-1, Nuclear transport, Carrier Proteins, Microtubule-Associated Proteins
Abstract

International audience; After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

Published
2015

15. Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

Author

Spencer L. Shorte, Juliette Fernandez, Anne Danckaert, Felipe Diaz-Griffero, Thomas Fricke, Pierre Charneau, Francesca Di Nunzio, Nathalie J. Arhel, Patricio Perez, Pascal Roux, and Emmanuelle Perret

Subjects
Science, Human immunodeficiency virus (HIV), lcsh:Medicine, Computational biology, Bioinformatics, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Medicine, Nuclear pore, lcsh:Science, Multidisciplinary, business.industry, lcsh:R, Correction, 3. Good health, Docking (molecular), 030220 oncology & carcinogenesis, lcsh:Q, Nucleoporin, Nuclear transport, business, 030217 neurology & neurosurgery
Abstract

There was an omission in the funding statement. Thomas Fricke (TF) was also supported by National Institutes of Health grant R01 AI087390. There was also an error in Author Contributions. All authors participated in the writing of the manuscript.

Published
2013

16. Residual HIV-1 DNA Flap-independent nuclear import of cPPT/CTS double mutant viruses does not support spreading infection

Author

Juliette Fernandez, Candela Iglesias, Francesca Di Nunzio, Nathalie J. Arhel, Mathieu Ringeard, Pierre Charneau, Philippe Souque, Raphael Gaudin, Centro de Investigacion en Enfermedades Infecciosas, Virologie moléculaire et vaccinologie, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Institut de génétique humaine ( IGH ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Immunité et cancer ( U932 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Immunité et cancer (U932), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and BMC, Ed.

Subjects
lcsh:Immunologic diseases. Allergy, Molecular Sequence Data, Active Transport, Cell Nucleus, HIV Infections, Biology, Virus Replication, medicine.disease_cause, Virus, Cell Line, 03 medical and health sciences, chemistry.chemical_compound, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Virology, medicine, Humans, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Mutation, Base Sequence, Research, 030302 biochemistry & molecular biology, Reverse Transcription, Molecular biology, Reverse transcriptase, 3. Good health, Cell nucleus, [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Infectious Diseases, medicine.anatomical_structure, Viral replication, chemistry, Cell culture, DNA, Viral, Codon, Terminator, HIV-1, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Nuclear transport, lcsh:RC581-607, DNA
Abstract

Background The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The central DNA Flap is a determinant of HIV-1 nuclear import, however, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. Therefore, to determine whether or not the DNA Flap is essential for HIV-1 nuclear import, we generated double mutant (DM) viruses, combining cPPT and CTS mutations to abolish DNA Flap formation. Results The combination of cPPT and CTS mutations reduced the proportion of viruses forming the central DNA Flap at the end of reverse transcription and further decreased virus infectivity in one-cycle titration assays. The most affected DM viruses were unable to establish a spreading infection in the highly permissive MT4 cell line, nor in human primary peripheral blood mononuclear cells (PBMCs), indicating that the DNA Flap is required for virus replication. Surprisingly, we found that DM viruses still maintained residual nuclear import levels, amounting to 5-15% of wild-type virus, as assessed by viral DNA circle quantification. Alu-PCR quantification of integrated viral genome also indicated 5-10% residual integration levels compared to wild-type virus. Conclusion This work establishes that the central DNA Flap is required for HIV-1 spreading infection but points to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication.

Published
2011

17. Cross-species amplification of 41 microsatellites in European cyprinids: a tool for evolutionary,population genetics and hybridization studies

Author

Emese Meglécz, Jean-François Martin, Melthide Sinama, Caroline Costedoat, Vincent Dubut, André Gilles, Juliette Fernandez, Rémi Chappaz, Université Paul Cézanne - Aix-Marseille 3, Centre de Biologie pour la Gestion des Populations (UMR CBGP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Institut de Recherche pour le Développement (IRD [France-Sud])-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut méditerranéen de biodiversité et d'écologie marine et continentale (IMBE), Avignon Université (AU)-Aix Marseille Université (AMU)-Institut de recherche pour le développement [IRD] : UMR237-Centre National de la Recherche Scientifique (CNRS), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Centre National de la Recherche Scientifique (CNRS)-Institut de recherche pour le développement [IRD] : UMR237-Aix Marseille Université (AMU)-Avignon Université (AU)

Subjects
0106 biological sciences, Scardinius, GENETIQUE DES POPULATIONS, [SDV]Life Sciences [q-bio], Short Report, lcsh:Medicine, Zoology, Population genetics, Toxostoma, 010603 evolutionary biology, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Leuciscus, cyprinide, Alburnoides bipunctatus, pcr, cyprinidae, lcsh:Science (General), lcsh:QH301-705.5, 030304 developmental biology, Medicine(all), évolution biologique, 0303 health sciences, [SDV.GEN.GPO]Life Sciences [q-bio]/Genetics/Populations and Evolution [q-bio.PE], biology, Squalius, Biochemistry, Genetics and Molecular Biology(all), Chondrostoma, lcsh:R, General Medicine, diversification des espèces, biology.organism_classification, lcsh:Biology (General), Chondrostoma toxostoma, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, europe, lcsh:Q1-390
Abstract

Background Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date. Findings Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species. Conclusions The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies.

Published
2010

18. HIV-1 capsid interacts with cytoskeletal-associated proteins for intracytoplasmic routing to the nucleus

Author

Spencer L. Shorte, Sandie Munier, Pierre Charneau, Anaba Zambo, Kathleen Gärtner, Yves Jacob, Anne Danckaert, Andreas Becker, Juliette Fernandez, Nathalie J. Arhel, Génétique et Ecologie des Virus, Génétique des Virus et Pathogénèse des Maladies Virales, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagopole (CITECH), Institut Pasteur [Paris], Virologie Structurale - Structural Virology, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Virologie moléculaire et vaccinologie, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and BMC, Ed.

Subjects
Viral protein, medicine.disease_cause, Bioinformatics, 03 medical and health sciences, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Virology, medicine, Nuclear pore, Nuclear membrane, Cytoskeleton, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Chemistry, 3. Good health, Cell biology, Infectious Diseases, medicine.anatomical_structure, Cytoplasm, Poster Presentation, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Nuclear transport, Host cytoskeleton, Nucleus, 030217 neurology & neurosurgery
Abstract

Background During infection, the Human Immunodeficiency Virus type 1 (HIV-1) uses the host cytoskeleton to traffic across the cytoplasm to the nucleus where its genome integrates into the host DNA. We have previously shown that HIV retrograde transport (i.e. towards the nucleus) results from the successive transfers from fast microtubule-directed movement to slower actinmediated movement closer to the nuclear compartment, resulting in docking at the nuclear pore [1]. However, neither the cellular cytoplasmic component(s) nor the viral protein(s) that interact to mediate transport have yet been identified. Recent data show that HIV-1 uncoating does not occur immediately after cell entry but near the nuclear membrane, suggesting that the viral structure interacting with the cytoskeleton during early retrograde trafficking might be the capsid.

Published
2013

19. Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

Author

Spencer L. Shorte, Pierre Charneau, Patricio Perez, Felipe Diaz-Griffero, Juliette Fernandez, Pascal Roux, Thomas Fricke, Anne Danckaert, Francesca Di Nunzio, Nathalie J. Arhel, Emmanuelle Perret, Virologie moléculaire et vaccinologie, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Imagopole (CITECH), Institut Pasteur [Paris], Albert Einstein College of Medicine [New York], This work was funded by the CNRS (Centre National de Recherche Scientifique), the ANRS (Agence Nationale de Recherche sur le SIDA), the FRM (Fondation pour la Recherche Médicale), the Sidaction, the Pasteur Institute and the European Union. ML, PP and FDG were supported by an National Institutes of Health R01 AI087390 grant and a pathway to independence award 4R00MH086162-02, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
Viral Diseases, [SDV]Life Sciences [q-bio], Cell Pores, lcsh:Medicine, HIV Infections, Chromosomal translocation, Plasma protein binding, MESH: HIV-1, Immunodeficiency Viruses, Molecular Cell Biology, Nuclear pore, lcsh:Science, 0303 health sciences, Multidisciplinary, 030302 biochemistry & molecular biology, Viral Replication Complex, MESH: HIV Infections, Cellular Structures, 3. Good health, Chromatin, Cell biology, Infectious Diseases, medicine.anatomical_structure, Medicine, Nucleoporin, MESH: Molecular Chaperones, MESH: Nuclear Pore, Research Article, Protein Binding, Active Transport, Cell Nucleus, MESH: Active Transport, Cell Nucleus, Viral Structure, Biology, Microbiology, Viral Preintegration Complex, 03 medical and health sciences, Virology, Viral Core, medicine, Humans, MESH: Protein Binding, Nuclear membrane, 030304 developmental biology, Cell Nucleus, MESH: Humans, lcsh:R, HIV, Viral Replication, Nuclear Pore Complex Proteins, MESH: HeLa Cells, HIV-1, Nuclear Pore, lcsh:Q, RANBP2, Nuclear transport, HeLa Cells, Molecular Chaperones, MESH: Nuclear Pore Complex Proteins
Abstract

International audience; The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

Published
2012

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