PB1-F2 Influenza A Virus Protein Adopts a β-Sheet Conformation and Forms Amyloid Fibers in Membrane Environments

• Published in: The Journal of biological chemistry Vol. 285; no. 17; pp. 13233 - 13243
• Main Authors: Chevalier, Christophe, Al Bazzal, Ali, Vidic, Jasmina, Février, Vincent, Bourdieu, Christiane, Bouguyon, Edwige, Le Goffic, Ronan, Vautherot, Jean-François, Bernard, Julie, Moudjou, Mohammed, Noinville, Sylvie, Chich, Jean-François, Da Costa, Bruno, Rezaei, Human, Delmas, Bernard
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23-04-2010; American Society for Biochemistry and Molecular Biology
• Subjects: Acetonitriles - chemistry; Amyloid - chemistry; Amyloid - genetics; Amyloid - metabolism; Amyloid - ultrastructure; Animals; Benzothiazoles; Biochemistry; Biochemistry, Molecular Biology; Cell Line; Cell Membrane - chemistry; Cell Membrane - genetics; Cell Membrane - metabolism; Diseases/Amyloid; Dogs; Humans; Influenza A Virus; Influenza A virus - chemistry; Influenza A virus - genetics; Influenza A virus - metabolism; Influenza A virus - pathogenicity; Life Sciences; Microbiology; Mutation; PB1-F2 Protein; Protein Structure and Folding; Protein Structure, Secondary; Protein/Folding; Protein/Structure; Protein/Synthesis; Protein/Viral; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thiazoles - chemistry; Trifluoroethanol - chemistry; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Viruses/Negative-strand RNA; Viruses/Negative-strand RNA; Influenza A Virus; Protein/Viral; Protein/Synthesis; Protein/Structure; PB1-F2 Protein; Protein/Folding; Diseases/Amyloid; ThT; TFE; dynamic light scattering; SPR; surface plasmon resonance; DLS; thioflavin T; ThS; IAV; influenza A virus; trifluoroethanol; thioflavin S
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.067710

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 α-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display β-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a β-sheet structure. Dynamic light scattering revealed that the presence of β-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.