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1. Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

Author

Léa Meyer, Isoline Duquénois, Stacy Gellenoncourt, Marie Pellerin, Aïlona Marcadet-Hauss, Nicole Pavio, and Virginie Doceul

Subjects
hepatitis E virus (HEV), interferon (IFN), IFN-stimulated gene (ISG), antiviral response, zoonosis, Immunologic diseases. Allergy, RC581-607
Abstract

IntroductionHepatitis E virus (HEV) is a common cause of enterically transmitted acute hepatitis worldwide. The virus is transmitted by the fecal-oral route via the consumption of contaminated water supplies and is also a zoonotic foodborne pathogen. Swine are the main reservoir of zoonotic HEV. In humans, HEV infection is usually asymptomatic or causes acute hepatitis that is self-limited. However, fulminant hepatic failure and chronic cases of HEV infection can occur in some patients. In contrast, HEV infection in pigs remains asymptomatic, although the virus replicates efficiently, suggesting that swine are able to control the virus pathogenesis. Upon viral infection, IFN is secreted and activates cellular pathways leading to the expression of many IFN-stimulated genes (ISGs). ISGs can restrict the replication of specific viruses and establish an antiviral state within infected and neighboring cells.MethodsIn this study, we used PCR arrays to determine the expression level of up to 168 ISGs and other IFN-related genes in the liver tissues of pigs infected with zoonotic HEV-3c and HEV-3f and in human bipotent liver HepaRG cells persistently infected with HEV-3f.Results and discussionThe expression of 12 and 25 ISGs was found to be up-regulated in infected swine livers and HepaRG cells, respectively. The expression of CXCL10, IFIT2, MX2, OASL and OAS2 was up-regulated in both species. Increased expression of IFI16 mRNA was also found in swine liver tissues. This study contributes to the identification of potential ISGs that could play a role in the control or persistence of HEV infection.

Published
2023
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2. Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry

Author

Mathieu Claireaux, Rémy Robinot, Jérôme Kervevan, Mandar Patgaonkar, Isabelle Staropoli, Anne Brelot, Alexandre Nouël, Stacy Gellenoncourt, Xian Tang, Mélanie Héry, Stevenn Volant, Emeline Perthame, Véronique Avettand-Fenoël, Julian Buchrieser, Thomas Cokelaer, Christiane Bouchier, Laurence Ma, Faroudy Boufassa, Samia Hendou, Valentina Libri, Milena Hasan, David Zucman, Pierre de Truchis, Olivier Schwartz, Olivier Lambotte, and Lisa A. Chakrabarti

Subjects
Science
Abstract

Here, Claireaux et al. show that people who naturally control HIV infection express lower levels of the viral co-receptor CCR5 in specific CD4+ T cells, and that this results from mutations or receptor internalization by CD4+ T cell-produced chemokines.

Published
2022
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3. Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential

Author

Bjoern Meyer, Jeanne Chiaravalli, Stacy Gellenoncourt, Philip Brownridge, Dominic P. Bryne, Leonard A. Daly, Arturas Grauslys, Marius Walter, Fabrice Agou, Lisa A. Chakrabarti, Charles S. Craik, Claire E. Eyers, Patrick A. Eyers, Yann Gambin, Andrew R. Jones, Emma Sierecki, Eric Verdin, Marco Vignuzzi, and Edward Emmott

Subjects
Science
Abstract

During SARS-CoV-2 replication, viral and cellular proteases play crucial roles and have been shown to be promising anti-viral targets. Here, Meyer et al. apply mass spectrometry to characterize the proteolytic cleavage profile of viral and cellular proteins in vitro.

Published
2021
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4. SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance

Author

Rémy Robinot, Mathieu Hubert, Guilherme Dias de Melo, Françoise Lazarini, Timothée Bruel, Nikaïa Smith, Sylvain Levallois, Florence Larrous, Julien Fernandes, Stacy Gellenoncourt, Stéphane Rigaud, Olivier Gorgette, Catherine Thouvenot, Céline Trébeau, Adeline Mallet, Guillaume Duménil, Samy Gobaa, Raphaël Etournay, Pierre-Marie Lledo, Marc Lecuit, Hervé Bourhy, Darragh Duffy, Vincent Michel, Olivier Schwartz, and Lisa A. Chakrabarti

Subjects
Science
Abstract

SARS-CoV-2 infection damages the airways. Here the authors show that SARS-CoV-2 infection induces the rapid loss of airway motile cilia, resulting in altered cilia clearance function. Cilia loss is preceded by reduced expression of the ciliogenesis regulator Foxj1.

Published
2021
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5. The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants

Author

Stacy Gellenoncourt, Nell Saunders, Rémy Robinot, Lucas Auguste, Maaran Michael Rajah, Jerôme Kervevan, Raphaël Jeger-Madiot, Isabelle Staropoli, Cyril Planchais, Hugo Mouquet, Julian Buchrieser, Olivier Schwartz, Lisa A. Chakrabarti, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université de Nîmes (UNIMES), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), This work was supported by the Urgence COVID-19 Fundraising Campaign of Institut Pasteur (COROCHIP, PFR4, and PFR7 projects) and Agence Nationale de Recherche sur le SIDA et les Maladies Infectieuses Emergentes (ANRS-MIE, project 19206) (L.A.C.) and by the Pasteur Institute, the European Health Emergency Preparedness and Response Authority (HERA), Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS-MIE, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR Coronamito, and IDISCOVR (O.S.). S.G. is the recipient of an MESR/Ecole Doctorale BioSPC, Université Paris Cité, fellowship, R.R. is the recipient of a Sidaction fellowship, and J.K. is the recipient of an ANRS fellowship, We thank Nathalie Aulner and the UTechS Photonic BioImaging (UPBI) core facility of Institut Pasteur, a member of the France BioImaging network, for training on the Opera imaging system. We thank Jacob Seeler for help with gel image acquisition and all the members of the Virus and Immunity Unit for reagents and helpful discussions. The following reagent was obtained through BEI Resources, NIAID, NIH: SARS-related coronavirus 2, Wuhan-Hu-1 spike-pseudotyped lentiviral kit V2, NR-53816., ANR-21-CO14-0007,CoronaMito,Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale(2021), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020)

Subjects
epistasis, variants, MESH: Humans, MESH: Mutation, SARS-CoV-2, [SDV]Life Sciences [q-bio], Immunology, COVID-19, proteolytic cleavage, MESH: Spike Glycoprotein, Coronavirus, spike, Microbiology, Virology, Insect Science, Mutation, Spike Glycoprotein, Coronavirus, Humans, MESH: COVID-19, MESH: SARS-CoV-2
Abstract

SARS-CoV-2 remained genetically stable during the first three months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide, and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes.AUTHOR SUMMARYThe successive emergence of SARS-CoV-2 variants is fueling the COVID pandemic, thus causing a major and persistent public health issue. The parameters involved in the emergence of variants with higher pathogenic potential remain incompletely understood. The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions, and resulting in viral particles with higher infectious capacity. The Alpha and the Delta variants that spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic when compared to the original SARS-CoV-2 strain. Interestingly, Alpha and Delta both carried mutations in a spike cleavage site that needs to be processed by cellular proteases prior to viral entry. The cleavage site mutations P681H/R made the Alpha and Delta spikes more efficient at viral fusion, by generating a higher fraction of cleaved spikes subunits S1 and S2. We show here that the early D614G mutation and the late P681H/R mutations act synergistically to increase the fusion capacity of SARS-CoV-2 variants. Specifically, viruses with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite to the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

Published
2022

6. SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation

Author

Mathieu Hubert, Jérémy Dufloo, Rémy Robinot, Hugo Mouquet, Olivier Schwartz, Ludivine Grzelak, Nell Saunders, Elodie Bishop, Françoise Porrot, Delphine Planas, Maaran Michael Rajah, Julian Buchrieser, Lisa A. Chakrabarti, Marija Zivaljic, Alice Bongers, Stacy Gellenoncourt, Florence Guivel-Benhassine, Cyril Planchais, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Vaccine Research Institute (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Sorbonne Université (SU), Neurobiologie intégrative des Systèmes cholinergiques / Integrative Neurobiology of Cholinergic Systems (NISC), Institut Pasteur [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Ecole doctorale Cerveau Cognition et Comportement [Paris] (ED 158 - 3C), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Work in OS lab is funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Fondation Pour la Recherche Médicale (FRM), Labex IBEID (ANR-10-LABX62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR CoronaMito AAP RA-COVID-19 V14, and IDISCOVR. Work in UPBI is funded by grant ANR-10-INSB-04-01 and Région Ile-de-France program DIM1-Health. MMR and MZ are supported by the Pasteur-Paris University (PPU) International Doctoral Program. MMR is also supported by Institut Pasteur Department of Virology 'Bourse de Soudure' fellowship. DP is supported by the Vaccine Research Institute. LG is supported by the French Ministry of Higher Education, Research and Innovation. EB is supported by the Medecine-Sciences ENS-PSL Program. HM laboratory is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69- 01), the INSERM, REACTing, EU (RECOVER), and Fondation de France (#00106077) grants., We thank members of the Virus and Immunity Unit for helpful discussions, Dr. Nicoletta Casartelli for her critical reading of the manuscript, and Nathalie Aulner and the UtechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis support., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-21-CO14-0007,CoronaMito,Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale(2021), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute [Créteil, France] (VRI), Institut Pasteur [Paris] (IP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Ziani, Isma, Laboratoires d'excellence - Initiative for the creation of a Vaccine Research Institute - - VRI2010 - ANR-10-LABX-0077 - LABX - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Protéomique du SARS-CoV-2 - - PROTEO-SARS-CoV-22020 - ANR-20-COVI-0059 - COVID-19 - VALID, Conséquences de l'infection par le SRAS-CoV-2 sur la fonction mitochondriale - - CoronaMito2021 - ANR-21-CO14-0007 - COVID-19 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, and Laboratoires d'excellence - GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE - - MILIEU INTERIEUR2010 - ANR-10-LABX-0069 - LABX - VALID

Subjects
fusion, coronavirus, MESH: Spike Glycoprotein, Coronavirus, MESH: Angiotensin-Converting Enzyme 2, medicine.disease_cause, Virus Replication, Giant Cells, SARS‐CoV‐2, MESH: Antibodies, Monoclonal, MESH: Giant Cells, MESH: Chlorocebus aethiops, Chlorocebus aethiops, MESH: Animals, Receptor, Coronavirus, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Syncytium, Strain (chemistry), General Neuroscience, Antibodies, Monoclonal, Articles, Transmembrane protein, Microbiology, Virology & Host Pathogen Interaction, Cell biology, MESH: HEK293 Cells, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Spike Glycoprotein, Coronavirus, MESH: Caco-2 Cells, Angiotensin-Converting Enzyme 2, MESH: Mutation, medicine.drug_class, Immunology, Alpha (ethology), MESH: Vero Cells, Biology, Monoclonal antibody, General Biochemistry, Genetics and Molecular Biology, Article, Cell Line, medicine, Animals, Humans, MESH: SARS-CoV-2, Beta (finance), syncytia, Molecular Biology, Vero Cells, MESH: Humans, General Immunology and Microbiology, SARS-CoV-2, MESH: Virus Replication, spike, MESH: Cell Line, HEK293 Cells, Mutation, Caco-2 Cells
Abstract

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation., Spike protein mutations expressed by emerging SARS‐CoV‐2 variants‐of‐concern differentially affect host cell‐to‐cell fusion, ACE2 receptor binding, and antibody escape.

Published
2021

7. Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential

Author

Jeanne Chiaravalli, Marius Walter, Edward Emmott, Emma Sierecki, Stacy Gellenoncourt, Philip Brownridge, Arturas Grauslys, Andrew R. Jones, Fabrice Agou, Charles S. Craik, Marco Vignuzzi, Yann Gambin, Patrick A. Eyers, Lisa A. Chakrabarti, Eric Verdin, Leonard A. Daly, Bjoern Meyer, Dominic P. Bryne, Claire E. Eyers, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Criblage chémogénomique et biologique (Plateforme) - Chemogenomic and Biological Screening Platform (PF-CCB), Institut Pasteur [Paris] (IP), Virus et Immunité - Virus and immunity (CNRS-UMR3569), University of Liverpool, Buck Institute for Research on Aging, University of California [San Francisco] (UC San Francisco), University of California (UC), University of New South Wales [Sydney] (UNSW), This work was supported by the Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant ANR-10-LABX-62-IBEID) to M.V. S.G. is the recipient of a MESR/Ecole Doctorale BioSPC ED562, Université de Paris fellowship. L.A.C. is supported by Institut Pasteur TASK FORCE SARS COV-2 (Tropicoro project), DIM ELICIT Region Ile-de-France, and ANRS. E.E. is supported by startup funding from the University of Liverpool, as well as a Wellcome Trust ISSF Interdisciplinary & Industry Award. E.E. is grateful for the support of GoFundMe donors for sponsoring SARS-CoV-2 research in his laboratory., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
Proteomics, MESH: Virus Internalization, viruses, medicine.medical_treatment, General Physics and Astronomy, Virus Replication, MESH: Dipeptides, MESH: RNA, Small Interfering, 2.2 Factors relating to the physical environment, MESH: COVID-19, MESH: Animals, MESH: Myosin-Light-Chain Kinase, RNA, Small Interfering, Aetiology, skin and connective tissue diseases, Lung, MESH: Protease Inhibitors, MESH: Viral Proteases, Multidisciplinary, medicine.diagnostic_test, Viral Proteases, MESH: Proteomics, Proteases, Dipeptides, src-Family Kinases, Infectious Diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Infection, Biotechnology, Proto-oncogene tyrosine-protein kinase Src, MESH: Antiviral Agents, MESH: Mutation, Science, Proteolysis, MESH: Proteolysis, Context (language use), Biology, Small Interfering, Antiviral Agents, Article, General Biochemistry, Genetics and Molecular Biology, Cell Line, Vaccine Related, Viral Proteins, Biodefense, medicine, Animals, Humans, Protease Inhibitors, MESH: SARS-CoV-2, Myosin-Light-Chain Kinase, MESH: Humans, Protease, SARS-CoV-2, Prevention, fungi, MESH: Virus Replication, COVID-19, MYLK, Pneumonia, General Chemistry, Virus Internalization, MESH: Viral Proteins, Virology, COVID-19 Drug Treatment, MESH: Cell Line, body regions, Emerging Infectious Diseases, Good Health and Well Being, MESH: src-Family Kinases, Viral replication, Mutation, RNA, Immunization
Abstract

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., During SARS-CoV-2 replication, viral and cellular proteases play crucial roles and have been shown to be promising anti-viral targets. Here, Meyer et al. apply mass spectrometry to characterize the proteolytic cleavage profile of viral and cellular proteins in vitro.

Published
2021

8. SARS-CoV-2 Alpha, Beta and Delta variants display enhanced Spike-mediated Syncytia Formation

Author

Florence Guivel-Benhassine, Lisa A. Chakrabarti, Mathieu Hubert, Julian Buchrieser, Nell Saunders, Maaran Michael Rajah, Ludivine Grzelak, Françoise Porrot, Hugo Mouquet, Cyril Planchais, Marija Zivaljic, Alice Bongers, Stacy Gellenoncourt, Rémy Robinot, Jérémy Dufloo, Elodie Bishop, Olivier Schwartz, and Delphine Planas

Subjects
Syncytium, Mutation, biology, Chemistry, medicine.drug_class, Alpha (ethology), medicine.disease_cause, Monoclonal antibody, Transmembrane protein, Cell biology, Interferon, medicine, biology.protein, Antibody, Beta (finance), medicine.drug
Abstract

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighbouring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha and Beta spread and fusion in cell cultures. Alpha and Beta replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Alpha, Beta and D614G fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes differentially modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.SynopsisThe Spike protein of the novel SARS-CoV-2 variants are comparative more fusogenic than the earlier strains. The mutations in the variant spike protein differential modulate syncytia formation, ACE2 binding, and antibody escape.The spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytia than D614GThe ACE2 affinity of the variant spike proteins correlates to their fusogenicityVariant associated mutations P681H, D1118H, and D215G augment cell-cell fusion, while antibody escape mutation E484K, K417N and Δ242-244 hamper it.Variant spike-mediated syncytia formation is effectively restricted by IFITMs

Published
2021

9. SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance

Author

Sylvain Levallois, Guillaume Duménil, Stéphane Rigaud, Françoise Lazarini, Olivier Gorgette, Lisa A. Chakrabarti, Rémy Robinot, Guilherme D. Melo, Julien Fernandes, Céline Trébeau, Pierre-Marie Lledo, Hervé Bourhy, Raphaël Etournay, Olivier Schwartz, Stacy Gellenoncourt, Samy Gobaa, Timothée Bruel, Florence Larrous, Mathieu Hubert, Marc Lecuit, Nikaïa Smith, Catherine Thouvenot, Darragh Duffy, Adeline Mallet, Vincent Michel, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Lyssavirus, épidémiologie et neuropathologie - Lyssavirus Epidemiology and Neuropathology, Institut Pasteur [Paris] (IP), Perception et Mémoire / Perception and Memory, Immunologie Translationnelle - Translational Immunology lab, Biologie des Infections - Biology of Infection, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Hub d'analyse d'images - Image Analysis Hub (Platform) (IAH), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut de l'Audition [Paris] (IDA), Plateforme technologique Biomatériaux et Microfluidique - Biomaterials and Microfluidics technologic Platform, Institut National de la Santé et de la Recherche Médicale (INSERM), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), This work was supported by : Institut Pasteur TASK FORCE SARS COV2 (Tropicoro project), DIM ELICIT Region Ile-de-France, and ANRS (L.A.C.), the Vaccine Research Institute (ANR-10-LABX-77), ANRS, Labex IBEID (ANR-10-LABX-62-IBEID), 'TIMTAMDEN' ANR-14-CE14-0029, 'CHIKV-Viro-Immuno' ANR-14-CE14- 0015-01, the Gilead HIV cure program, ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR (O.S.), Institut Pasteur TASK FORCE SARS COV2 and ANR Flash Covid CoVarImm (D.D.), Institut Pasteur TASK FORCE SARS COV2 (Neuro-Covid project) (H.B.). The Lledo’s lab is supported by the life insurance company 'AG2R-La-Mondiale'. The UtechS Photonic BioImaging (Imagopole) and the UtechS Ultrastructural BioImaging (UBI) are supported by the French National Research Agency (France BioImaging, ANR-10–INSB–04, Investments for the Future). R.R. is the recipient of a Sidaction fellowship, N.S. of a Pasteur-Roux Cantarini fellowship, and St.G. of a MESR/Ecole Doctorale B3MI, Paris 7 University fellowship. S.L. is supported by FRM (fellowship ECO201906009119) and by 'Ecole Doctorale FIRE – Programme Bettencourt'., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-20-COVI-0053,CoVarImm,Variation de la réponse immune systémique et muqueuse pendant l'infection par le SRAS-CoV-2 et la convalescence(2020), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Vaccine Research Institute (VRI), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Axoneme, 0303 health sciences, Tight junction, Mucociliary clearance, Cilium, Biology, respiratory system, 3. Good health, Cell biology, respiratory tract diseases, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Parenchyma, Motile cilium, medicine, Basal body, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030304 developmental biology, Respiratory tract
Abstract

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile ciliain vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Published
2021

10. In vitro impact of amino acid-derived bacterial metabolites on colonocyte mitochondrial activity, oxidative stress response and DNA integrity

Author

François Blachier, Vincent Bitane, Mireille Andriamihaja, Stacy Gellenoncourt, Annaïg Lan, Lucie Armand, Physiologie de la Nutrition et du Comportement Alimentaire (PNCA), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, and European Project: 613979,EC:FP7:KBBE,FP7-KBBE-2013-7-single-stage,MYNEWGUT(2013)

Subjects
0301 basic medicine, Colon, Cellular respiration, DNA damage, [SDV]Life Sciences [q-bio], Biophysics, In Vitro Techniques, medicine.disease_cause, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Oxygen Consumption, medicine, Humans, Amino Acids, Molecular Biology, Colonocytes, chemistry.chemical_classification, Bacterial metabolites, Reactive oxygen species, Bacteria, 030102 biochemistry & molecular biology, Acetaldehyde, DNA, biochemical phenomena, metabolism, and nutrition, Amino acid, Cell respiration, Comet assay, Oxidative Stress, 030104 developmental biology, chemistry, [SDV.TOX]Life Sciences [q-bio]/Toxicology, HT29 Cells, Genotoxicity, Oxidative stress
Abstract

Background 4-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with cytotoxicity, urging us to test HO-PAA's effects on human colonocytes. We compared these effects with those of phenylacetic acid (PAA), phenol and acetaldehyde, also issued from amino acids fermentation. Methods HT-29 Glc−/+ human colonocytes were exposed for 24 h to metabolites at concentrations between 350 and 1000 μM for HO-PAA and PAA, 250-1500 μM for phenol and 25-500 μM for acetaldehyde. We evaluated metabolites'cytotoxicity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and DNA quantification assays, reactive oxygen species (ROS) production with H2DCF-DA, and DNA damage with the comet assay. We measured cell oxygen consumption and mitochondrial complexes activity by polarography. Results Although HO-PAA displayed no cytotoxic effect on colonocytes, it decreased mitochondrial complex I activity and oxygen consumption. This was paralleled by an increase in ROS production and DNA alteration. Cells pretreatment with N-acetylcysteine, a ROS scavenger, decreased genotoxic effects of HO-PAA, indicating implication of oxidative stress in HO-PAA's genotoxicity. PAA and phenol did not reproduce these effects, but were cytotoxic towards colonocytes. Last, acetaldehyde displayed no effect in terms of cytotoxicity and mitochondrial metabolic activity, but increased DNA damage. Conclusions Several bacterial metabolites produced from amino acids displayed deleterious effects on human colonocytes, in terms of genotoxicity (HO-PAA and acetaldehyde) or cytotoxicity (PAA and phenol). General significance This study helps understanding the consequences of intestinal microbiota's metabolic activity on the host since amino acids fermentation can lead to the formation of compounds toxic towards colonic epithelial cells.

Published
2019

11. Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry

Author

Mathieu Claireaux, Rémy Robinot, Jérôme Kervevan, Mandar Patgaonkar, Isabelle Staropoli, Anne Brelot, Alexandre Nouël, Stacy Gellenoncourt, Xian Tang, Mélanie Héry, Stevenn Volant, Emeline Perthame, Véronique Avettand-Fenoël, Julian Buchrieser, Thomas Cokelaer, Christiane Bouchier, Laurence Ma, Faroudy Boufassa, Samia Hendou, Valentina Libri, Milena Hasan, David Zucman, Pierre de Truchis, Olivier Schwartz, Olivier Lambotte, Lisa A. Chakrabarti, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Laboratoire de Microbiologie Clinique [AP-HP Hôpital Necker-Enfants Malades], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biomics (plateforme technologique), Centre de recherche en épidémiologie et santé des populations (CESP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Hôpital Foch [Suresnes], Hôpital Raymond Poincaré [AP-HP], 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), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Université Paris-Sud - Paris 11 (UP11), This work was supported by grants to L.A.C. from ANRS (17218 and 19206), Institut Pasteur (PasteurInnov TETRHIS), and ANR (14 CE 16002901). The Biomics Platform is a member of the 'France Génomique' consortium (ANR10-INBS-09-08). M.C. was the recipient of a Sidaction fellowship, R.R. of a Pasteur Carnot MS and a Sidaction fellowships, M.P. of an ANRS fellowship, and S.G. of a MESR/Université de Paris fellowship., We are grateful to patients who participated in the study. We thank the investigators of the ANRS CO21 CODEX cohort and Camille Lecuroux for help with recruitment of controller patients, Morgane Marcou and Katia Bourdic for help with recruitment of treated patients, Bernard Lagane for the gift of plamids, and Sophie Novault from the Pasteur CB UTechS for advice on cell sorting. MHC class II tetramer reagents were provided by the NIH Tetramer Core facility at Emory University. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 PTE Gag peptide set (Cat # 11554)., ANR-14-CE16-0029,PD1VAX,Une nouvelle stratégie de vaccination par vecteur ADN PD1 pour mimer les réponses spécifiques de Gag trouvées chez les contrôleurs spontanés du VIH(2014), and ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010)

Subjects
CD4-Positive T-Lymphocytes, Viral membrane fusion, MESH: Mutation, Receptors, CXCR3, Receptors, CCR5, Science, MESH: Virus Internalization, viruses, General Physics and Astronomy, Down-Regulation, Gene Products, gag, HIV Infections, MESH: Receptors, CCR5, MESH: Down-Regulation, General Biochemistry, Genetics and Molecular Biology, Article, MESH: Receptors, CXCR3, MESH: HIV-1, MESH: Elite Controllers, Humans, MESH: Humans, Multidisciplinary, Retrovirus, Histocompatibility Antigens Class II, MESH: CD4-Positive T-Lymphocytes, virus diseases, MESH: HIV Infections, General Chemistry, Virus Internalization, MESH: Chemokines, MESH: Gene Expression Regulation, Gene Expression Regulation, MESH: Gene Products, gag, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Mutation, MESH: Histocompatibility Antigens Class II, HIV-1, Chemokines, Elite Controllers
Abstract

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of β-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control., Here, Claireaux et al. show that people who naturally control HIV infection express lower levels of the viral co-receptor CCR5 in specific CD4+ T cells, and that this results from mutations or receptor internalization by CD4+ T cell-produced chemokines.

Published
2019

12. Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation

Author

Marc Delarue, Catherine Van Renterghem, Zaineb Fourati, Pierre-Jean Corringer, Ludovic Sauguet, Haidai Hu, Ákos Nemecz, Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), Récepteurs Canaux - Channel Receptors, Á.N. and Z.F. were supported by Agence Nationale de Recherches Grant 13-BSV8-0020 'Pentagate.' H.H. was sponsored by the China Scholarship Council and Institut Pasteur., We thank Anaïs Menny for sharing expertise on plasmid constructions, the staff of the Crystallography Platform (PF6) in the Institut Pasteur for initial crystal screens, Reinis Reinholds Ruza for help in data collection, Jérôme Loc’h for help during structural refinement and picture preparation, Stacy Gellenoncourt for help in purification and crystallization of the sTeLIC R86A mutant, the staff of synchrotron beamlines in Soleil (Orsay) and European Synchrotron Radiation Facility (Grenoble) for data collection, Pierre Legrand (Soleil synchrotron) for suggestions on refinement in Buster, and Marc Gielen for suggestions during the paper preparation, ANR-13-BSV8-0020,Pentagate,Mécanismes d'activation et de désensibilisation d'un récepteur-canal pentamérique(2013), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Gating, Crystallography, X-Ray, Divalent, 03 medical and health sciences, chemistry.chemical_compound, Allosteric Regulation, Bacterial Proteins, [CHIM.CRIS]Chemical Sciences/Cristallography, structural biology, Binding site, crystallography, Ion channel, Tetramethylammonium, chemistry.chemical_classification, Multidisciplinary, ligand-gated ion channel, druggable cavity, Hydrogen-Ion Concentration, Ligand-Gated Ion Channels, electrophysiology, Transmembrane protein, Quaternary Ammonium Compounds, 030104 developmental biology, chemistry, Structural biology, PNAS Plus, Biophysics, Ligand-gated ion channel, Gammaproteobacteria
Abstract

International audience; Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Published
2018

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