New insights into the understanding of hepatitis C virus entry and cell-to-cell transmission by using the ionophore Monensin A

Autor: Sandrine Belouzard, Dimitri Lavillette, Didier Hober, Laurence Cocquerel, Yves Rouillé, Florian Douam, Noémie Calland, Thibaut Vausselin, Famara Sane, C. Wychowski, Thomas F. Baumert, Julie Potel, Jean Dubuisson, Véronique Descamps, Lucie Fénéant, Gilles Duverlie, Catherine François
Přispěvatelé: Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - 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 National de la Recherche Scientifique (CNRS), Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), 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), This work was supported by the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS). L.F., N.C., and T.V. were supported by a fellowship from ANRS. L.F., J.P., N.C., and T.V. were supported by a fellowship from the French Ministry of Research. S.B. was supported by a Marie Curie International Reintegration Grant (grant PIRG-GA-2009-256300)., We thank S. Ung and A. Pillez for their technical assistance. We are grateful to S. Levy, J. McKeating, E. Rubinstein, T. Pietschmann, J. Bukh, T. Wakita, J. Ball, M. MacDonald, J.-W. Yoon, and J. McFarlane for providing us with reagents. We thank the BioImaging Center Lille-Nord de France for access to instruments., Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut National de la Recherche Agronomique (INRA)-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), 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), 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)
Jazyk: angličtina
Rok vydání: 2015
Předmět:
[SDV]Life Sciences [q-bio]
Mutant
Hepacivirus
medicine.disease_cause
MESH: Dose-Response Relationship
Drug
chemistry.chemical_compound
MESH: Virus Internalization/drug effects
Viral Envelope Proteins
MESH: Viral Proteins/metabolism
Fluorescent Antibody Technique
Indirect
Mutation
biology
Monensin
MESH: Neutralization Tests
virus diseases
Hydrogen-Ion Concentration
3. Good health
Virus-Cell Interactions
MESH: Viral Proteins/genetics
MESH: Ionophores/pharmacology
MESH: Mutation
Missense/genetics
MESH: Viral Envelope Proteins/genetics
MESH: Hepacivirus/genetics
Intracellular pH
Immunology
Mutation
Missense
MESH: Drug Resistance
Viral/genetics
Microbiology
Clathrin
Virus
Cell Line
Viral Proteins
Neutralization Tests
Virology
Drug Resistance
Viral
medicine
Humans
MESH: Fluorescent Antibody Technique
Indirect
MESH: Humans
Dose-Response Relationship
Drug
Ionophores
MESH: Hydrogen-Ion Concentration/drug effects
Virus Internalization
Molecular biology
digestive system diseases
Hypervariable region
MESH: Cell Line
chemistry
MESH: Hepacivirus/physiology
Cell culture
MESH: Monensin/pharmacology
Insect Science
biology.protein
Zdroj: Journal of Virology
Journal of Virology, American Society for Microbiology, 2015, 89 (16), pp.8346-64. ⟨10.1128/JVI.00192-15⟩
Journal of Virology, 2015, 89 (16), pp.8346-64. ⟨10.1128/JVI.00192-15⟩
ISSN: 0022-538X
1098-5514
DOI: 10.1128/JVI.00192-15⟩
Popis: In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCE Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.
Databáze: OpenAIRE
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