title: New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Fénéant
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Potel
authors.affiliation: Virology Department, EA4294 UPJV, Amiens University Hospital, Amiens, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: François
authors.affiliation: Laboratoire de Virologie EA3610, Université Lille 2, CHRU Lille, Lille, France.
authors.forename: Famara
authors.initials: F
authors.lastname: Sané
authors.affiliation: CNRS-UMR5557, Microbial Ecology, Université Claude Bernard Lyon 1, Villeurbanne, France.
authors.forename: Florian
authors.initials: F
authors.lastname: Douam
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Belouzard
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Noémie
authors.initials: N
authors.lastname: Calland
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Vausselin
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Rouillé
authors.affiliation: Virology Department, EA4294 UPJV, Amiens University Hospital, Amiens, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Descamps
authors.affiliation: INSERM U1110, Université de Strasbourg, Pôle Hépato-Digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Baumert
authors.affiliation: Virology Department, EA4294 UPJV, Amiens University Hospital, Amiens, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.affiliation: CNRS-UMR5557, Microbial Ecology, Université Claude Bernard Lyon 1, Villeurbanne, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Lavillette
authors.affiliation: Laboratoire de Virologie EA3610, Université Lille 2, CHRU Lille, Lille, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Hober
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.affiliation: Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France laurence.cocquerel@ibl.cnrs.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Cocquerel
mesh_terms: D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology*; D008985:Monensin / Q000494:pharmacology*; D020125:Mutation, Missense / Q000235:genetics; D009500:Neutralization Tests; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: C087035:E1 protein, Hepatitis C virus; C400562:HVR1 protein, Hepatitis C virus; D007476:Ionophores; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C091015:glycoprotein E2, Hepatitis C virus; D008985:Monensin