PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.
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.
Chevalier
Christophe
C
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
Al Bazzal
Ali
A
Vidic
Jasmina
J
Février
Vincent
V
Bourdieu
Christiane
C
Bouguyon
Edwige
E
Le Goffic
Ronan
R
Vautherot
Jean-François
JF
Bernard
Julie
J
Moudjou
Mohammed
M
Noinville
Sylvie
S
Chich
Jean-François
JF
Da Costa
Bruno
B
Rezaei
Human
H
Delmas
Bernard
B
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
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