title: Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads.
abstract: Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1-E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1-E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1-E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.
authors.affiliation: CBMN, UMR CNRS 5248, Université Bordeaux 1, ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bonnafous
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR CNRS 5086, Université Lyon 1, IFR128 Lyon Biosciences Gerland, F-69007 Lyon, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Perrault
authors.affiliation: CBMN, UMR CNRS 5248, Université Bordeaux 1, ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Le Bihan
authors.affiliation: INSERM, U758, F-69007 Lyon, France.|Université de Lyon, UCB-Lyon 1, IFR128, F-69007 Lyon, France.|Ecole Normale Supérieure de Lyon, F-69007 Lyon, France.
authors.forename: Birke
authors.initials: B
authors.lastname: Bartosch
authors.affiliation: Ecole Normale Supérieure de Lyon, F-69007 Lyon, France.|INSERM, U758, F-69007 Lyon, France.|Université de Lyon, UCB-Lyon 1, IFR128, F-69007 Lyon, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Lavillette
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR CNRS 5086, Université Lyon 1, IFR128 Lyon Biosciences Gerland, F-69007 Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: CBMN, UMR CNRS 5248, Université Bordeaux 1, ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Lambert
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR CNRS 5086, Université Lyon 1, IFR128 Lyon Biosciences Gerland, F-69007 Lyon, France.
authors.forename: Eve-Isabelle
authors.initials: EI
authors.lastname: Pécheur
mesh_terms: D000914:Antibodies, Viral / Q000378:metabolism; D020285:Cryoelectron Microscopy; D052203:Ferrosoferric Oxide; D016174:Hepacivirus / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure*; D008081:Liposomes; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000378:metabolism; D014771:Virion / Q000302:isolation & purification / Q000648:ultrastructure; D022701:Virosomes / Q000302:isolation & purification; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D000914:Antibodies, Viral; C087035:E1 protein, Hepatitis C virus; D008081:Liposomes; D014759:Viral Envelope Proteins; D022701:Virosomes; C091015:glycoprotein E2, Hepatitis C virus; D052203:Ferrosoferric Oxide