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

Synchrotron Infrared and Deep UV Fluorescent Microspectroscopy Study of PB1-F2 β-Aggregated Structures in Influenza A Virus-infected Cells

“…No labels, stains, or dyes are required for FTIR microspectroscopy and it is a non-destructive, sensitive and fast tool. We had previously observed the presence of ThSpositive aggregates in IAV-infected monocytic cells (25). However, we failed to detect a specific ThS positive signal by fluorescent microscopy in WT-infected A549 cells although we proved that oligomerized forms of PB1-F2 were present in such cells using others methods (23,28).…”

“…Generation of Recombinant Knocked Out PB1-F2 Mutant Influenza Virus-Influenza A/WSN/1933 (H1N1) wild-type (WT) and the PB1-F2 knock-out (F2) viruses used in this study were produced using the 12-plasmid reverse genetic system as previously described (25). Briefly, 293T cells were transfected with 12 plasmids following the FuGENE protocol (Roche): eight plasmids encoding the viral RNA segments of IAV and four encoding the three subunits of the viral polymerase and the nucleoprotein.…”

“…Moreover, PB1-F2 has a strong propensity to oligomerize. We previously showed by thioflavin staining that PB1-F2 formed amyloid fibers in vitro using recombinant protein and in IAV-infected cells (25). PB1-F2 is present as unstructured monomers in the early stage of infection (23,26,27), and rapidly forms soluble ␤-oligomers (28,29) to finally accumulate as ␤-amyloid aggregates at a later stage of infection (25).…”

“…PB1-F2 is disordered in aqueous solution (24) but can switch from a random state to a ␤-sheet secondary structure in a membrane-mimicking environment (25). Moreover, PB1-F2 has a strong propensity to oligomerize.…”

“…We previously showed by thioflavin staining that PB1-F2 formed amyloid fibers in vitro using recombinant protein and in IAV-infected cells (25). PB1-F2 is present as unstructured monomers in the early stage of infection (23,26,27), and rapidly forms soluble ␤-oligomers (28,29) to finally accumulate as ␤-amyloid aggregates at a later stage of infection (25). Interestingly, other reports referred to the propensity of PB1-F2 to oligomerize and to be implicated in the extracellular activation of the NLRP3 inflammasome pathway (30), which is known to be triggered by protein amyloid aggregates (31).…”