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.

FIGURE 1.

A, multialignment of the seven PB1-F2 variants included in this study. Strains are abbreviated as follows. 1918, A/Brevig Mission/1/1918 (H1N1); WSN, A/WSN/1933 (H1N1); PR8, A/PR/8/1934 (H1N1); UD72, A/Udorn/1972 (H3N2); HK97, A/HK/156/1997 (H5N1); FR06, A/Swan/FR/06299/2006 (H5N1); MA65, A/Tu/Mass/3740/1965 (H6N2). Conserved residues for the seven sequences are indicated on the bottom line (blue, charged residues; red, hydrophobic residues). B, secondary structure predictions (blue boxes, α-helix; red boxes, β-turn) were obtained with the software GOR-IV (available on the World Wide Web). C, the left panel shows SDS-PAGE analysis and Coomassie Blue staining of purified PB1-F2 proteins. The relative molecular weights (shown in thousands) were determined by reference to marker proteins. D, far-UV CD spectra for WSN PB1-F2 recorded in 10 mm sodium acetate, pH 5, buffer. E, the right panel shows the evolution in time (24 h) of the mean R_H of WSN PB1-F2 (50 μm) in 10 mm sodium acetate, pH 5, buffer measured by DLS at 20 °C.

FIGURE 2.

Secondary structure formation of the seven PB1-F2 variants in different environments. Far-UV CD spectra were recorded in 10 mm sodium acetate, pH 5, buffer complemented with solvants (50% TFE (A) or 80% acetonitrile (C)), detergent (0.01% SDS (D)), or 0.1 mg/ml asolectin liposomes (E). B, the secondary structural switch from random coil to β-sheet conformation while increasing acetonitrile percentage in the buffer for FRO6 PB1-F2.

FIGURE 3.

DLS size distributions of the mean R_H, of different PB1-F2 variants at a concentration of 50 μm in 10 mm sodium acetate, pH 5, buffer alone (A) or complemented with 50% TFE (B) or 80% acetonitrile (C). The inset in C shows aggregates formed by WSN PB1-F2 in acetonitrile by electron microscopy. D, evolution of the R_H of WSN and FRO6 PB1-F2 in the presence of 0.01% (red), 0.1% (green), or 1% (blue) SDS. Observation by electron microscopy of the fibers (E) formed by WSN (left) and FRO6 (right) PB1-F2 variants in the presence of 0.01% SDS. Bars, 200 nm.

FIGURE 4.

A, measure of the fluorescence increase of ThT after binding with amyloid formed by the seven PB1-F2 variants at a final concentration of 5 μm in 10 mm sodium acetate pH5 buffer containing 0.01% SDS. B, ThT binding by 1918 PB1-F2 at concentrations ranging from 0.25 to 5 μm. The control was obtained with PB1-F2 at the same concentration in sodium acetate buffer without SDS. AU, absorbance units.

FIGURE 5.

Membrane permeabilization by PB1-F2 variants. Asolectin liposomes were assayed for calcein release against nanomolar concentrations of PB1-F2 variants. A, permeabilization of liposomes upon the addition of 12.5–500 nm FRO6 PB1-F2 variant in 10 mm sodium acetate, pH 5. B, lysis activity of all seven PB1-F2 variants at a concentration of 25 nm in 10 mm sodium acetate, pH 5, buffer. C, images of liposomes in the presence of different PB1-F2 variants by electron microscopy. Left, untreated liposomes in sodium acetate buffer, pH 5; upper right, WSN PB1-F2; lower right, FRO6 PB1-F2. Bar, 200 nm.

FIGURE 6.

PB1-F2 expression and aggregation in influenza virus-infected cells. A, real-time kinetics of interaction between lysates obtained from U937-infected cells with wild type A/WSN/1933 (H1N1) virus (WT), virus knocked out for PB1-F2 expression (ΔPB1-F2), or non-infected cells with chip-immobilized monoclonal anti-PB1-F2 antibody. The sensorgrams are representative for three independent experiments. Inset, a calibration curve obtained for interaction of purified WSN PB1-F2 with the immobilized anti-PB1-F2 antibody. B, ThS staining of nonstimulated (NS), WT, and ΔPB1-F2 U937-infected cells was observed by fluorescence microscopy after excitation with UV light. Bar, 10 μm.

FIGURE 7.

Model for the PB1-F2 folding pathways. The scheme represents the different folding pathways leading to the formation of either disorder conformations, α-helices, or oligomeric β-enriched structures, depending on the physical and chemical parameters of the environment. All PB1-F2 variants exhibit random structures in aqueous (acidic pH) solutions. PB1-F2 variants adopt an α-helical structure in TFE-containing solutions. Whereas ThT-negative β-sheet assemblies are evidenced in acetonitrile solutions, PB1-F2 variants generate ThT positive amyloidic assemblies in the presence of 0.01% SDS. The length of the fibers differs, depending on the PB1-F2 variants. β-Oligomers are also observed in the presence of liposomes.