title: PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.
abstract: The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.
authors.affiliation: Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, UR892, Domaine de Vilvert, F-78350 Centre de Jouy-en-Josas, France. christophe.chevalier@jouy.inra.fr
authors.forename: Christophe
authors.initials: C
authors.lastname: Chevalier
authors.forename: Ali
authors.initials: A
authors.lastname: Al Bazzal
authors.forename: Jasmina
authors.initials: J
authors.lastname: Vidic
authors.forename: Vincent
authors.initials: V
authors.lastname: Février
authors.forename: Christiane
authors.initials: C
authors.lastname: Bourdieu
authors.forename: Edwige
authors.initials: E
authors.lastname: Bouguyon
authors.forename: Ronan
authors.initials: R
authors.lastname: Le Goffic
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Vautherot
authors.forename: Julie
authors.initials: J
authors.lastname: Bernard
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moudjou
authors.forename: Sylvie
authors.initials: S
authors.lastname: Noinville
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Chich
authors.forename: Bruno
authors.initials: B
authors.lastname: Da Costa
authors.forename: Human
authors.initials: H
authors.lastname: Rezaei
authors.forename: Bernard
authors.initials: B
authors.lastname: Delmas
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D009154:Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000097:Acetonitriles; D000682:Amyloid; D052160:Benzothiazoles; C442816:PB1-F2 protein, Influenza A virus; D011994:Recombinant Proteins; D013844:Thiazoles; D014764:Viral Proteins; C009462:thioflavin T; D014270:Trifluoroethanol; C032159:acetonitrile