title: Identification of conserved residues in hepatitis C virus envelope glycoprotein E2 that modulate virus dependence on CD81 and SRB1 entry factors.
abstract: In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.
  
  
  OBJECTIVE
  Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.
authors.forename: Muriel
authors.initials: M
authors.lastname: Lavie
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR-5086-CNRS, Labex Ecofect, Université de Lyon, Lyon, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Sarrazin
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR-5086-CNRS, Labex Ecofect, Université de Lyon, Lyon, France.
authors.forename: Roland
authors.initials: R
authors.lastname: Montserret
authors.affiliation: Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, 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: Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie Jules Verne, Amiens, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.
authors.forename: Karin
authors.initials: K
authors.lastname: Séron
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, UMR-5086-CNRS, Labex Ecofect, Université de Lyon, Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France jean.dubuisson@ibl.cnrs.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D051122:Scavenger Receptors, Class B / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D060225:Tetraspanin 28 / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D011991:Receptors, Virus; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus