title: Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion.
abstract: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein.
  
  
  OBJECTIVE
  HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, LabEx Ecofect, University of Lyon, Lyon, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.affiliation: CIRI-International Center for Infectiology Research, INSERM U1111, Ecole Normale Supérieure, CNRS UMR 5308, LabEx Ecofect, University of Lyon, Lyon, France.
authors.forename: Birke
authors.initials: B
authors.lastname: Bartosch
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France.
authors.forename: Khaled
authors.initials: K
authors.lastname: Alsaleh
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France.
authors.forename: Birke Andrea
authors.initials: BA
authors.lastname: Tews
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, LabEx Ecofect, University of Lyon, Lyon, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Loquet
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Ciczora
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Riva
authors.affiliation: Laboratoire des Protéines et Systèmes Membranaires, UMR 9198, Institut de Biologie Intégrative de la Cellule (I2BC), CEA, CNRS and Université Paris-Sud, CEA Saclay, Gif-sur-Yvette, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Montigny
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Montpellier
authors.affiliation: Laboratoire de Virologie EA4294, Centre Hospitalier Universitaire d'Amiens, Université de Picardie, Amiens, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, LabEx Ecofect, University of Lyon, Lyon, France.
authors.forename: Eve-Isabelle
authors.initials: EI
authors.lastname: Pécheur
authors.affiliation: Laboratoire des Protéines et Systèmes Membranaires, UMR 9198, Institut de Biologie Intégrative de la Cellule (I2BC), CEA, CNRS and Université Paris-Sud, CEA Saclay, Gif-sur-Yvette, France.
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.affiliation: CIRI-International Center for Infectiology Research, INSERM U1111, Ecole Normale Supérieure, CNRS UMR 5308, LabEx Ecofect, University of Lyon, Lyon, France.
authors.forename: François-Loïc
authors.initials: FL
authors.lastname: Cosset
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U1019, Institut Pasteur de Lille, University of Lille, Lille, France jean.dubuisson@ibl.cnrs.fr f.penin@ibcp.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, LabEx Ecofect, University of Lyon, Lyon, France jean.dubuisson@ibl.cnrs.fr f.penin@ibcp.fr.
authors.forename: François
authors.initials: F
authors.lastname: Penin
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics; D014771:Virion / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D019065:Virus Assembly; D053586:Virus Internalization
chemical_list: C087035:E1 protein, Hepatitis C virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus