title: Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity.
abstract: PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas, jasmina.vidic@jouy.inra.fr.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Vidic
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas.
authors.forename: Charles-Adrien
authors.initials: CA
authors.lastname: Richard
authors.affiliation: the Génétique Animale et Biologie Intégrative, INRA, UMR1313, Domaine de Vilvert, 78350 Jouy en Josas, and.
authors.forename: Christine
authors.initials: C
authors.lastname: Péchoux
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas.
authors.forename: Bruno
authors.initials: B
authors.lastname: Da Costa
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bertho
authors.affiliation: the Institut de Chimie Moléculaire et des Matériaux d'Orsay, Université Paris-Sud, CNRS, UMR 8182, 91400 Orsay, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Mazerat
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas.
authors.forename: Bernard
authors.initials: B
authors.lastname: Delmas
authors.affiliation: From the Unité de Virologie et Immunologie Moléculaires, INRA, UR892, Domaine de Vilvert, 78350 Jouy en Josas.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chevalier
mesh_terms: D000682:Amyloid / Q000633:toxicity*; D000890:Anti-Infective Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000472:pathogenicity / Q000648:ultrastructure; D008081:Liposomes / Q000378:metabolism* / Q000648:ultrastructure; D008826:Microbial Sensitivity Tests; D010539:Permeability; D066329:Protein Aggregates / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000633:toxicity*
chemical_list: D000682:Amyloid; D000890:Anti-Infective Agents; D008081:Liposomes; C442816:PB1-F2 protein, Influenza A virus; D066329:Protein Aggregates; D014764:Viral Proteins
keywords: amyloid; cell permeabilization; influenza virus; intrinsically disordered protein; liposome; viral protein