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PB1-F2 is a non-structural protein of influenza A viruses (IAV) that modulates viral pathogenesis in a host-specific manner. In mammals, this protein has been shown to increase IAV virulence by delaying the early immune response and, eventually, exacerbating lung inflammation at the late stage of infection. PB1-F2 is a small protein, but displays very high sequence polymorphism and sequence length disparity depending on viral strain. These features result in strong variations in the cellular activity of PB1-F2. Studies have also reported that the effect of PB1-F2 is cell-type dependent. It has notably been shown that PB1-F2 can promote apoptosis in immune cells, but not in epithelial cells. This phenomenon appears to be partly related to the higher order structure of the protein, given that the presence of PB1-F2 β-aggregated structures in infected immune cells correlates with cell death induction. In this work, we evaluated, by synchrotron Fourier transform infrared spectromicroscopy, the impact of the transient expression of PB1-F2 on the biochemical composition of the human epithelial cell line HEK293T. Two PB1-F2 variants that are closely related to each other but derived from a strain with high [A/BrevigMission/1/1918 (H1N1)] or a low [A/WSN/1933 (H1N1)] virulence were studied here. Infrared spectra analysis revealed no specific enrichment of β-aggregated structures in PB1-F2-expressing cells. Nevertheless, this analysis suggested that there is a higher content of β-sheet secondary structures in the PB1-F2 from A/WSN/1933 than that from A/BrevigMission/1/1918. Our data also showed no change in membrane composition in the presence of PB1-F2, implying that PB1-F2 does not promote apoptosis in HEK293T cells. Finally, we found that the PB1-F2 from A/WSN/1933 interferes with adenosine triphosphate production, suggesting that this PB1-F2 variant may disturb the mitochondrial activity.

PB1-F2 is a virulence factor of influenza A virus (IAV) whose functions remain misunderstood. The different roles of PB1-F2 may be linked to its structural polymorphism and to its propensity to assemble into oligomers and amyloid fibers in the vicinity of the membrane of IAV-infected cells. Here, we monitored the impact of PB1-F2 on the biochemical composition and protein structures of human epithelial pulmonary cells (A549) and monocytic cells (U937) upon IAV infection using synchrotron Fourier-transform infrared (FTIR) and deep UV (DUV) microscopies at the single-cell level. Cells were infected with a wild-type IAV and its PB1-F2 knock-out mutant for analyses at different times post-infection. IR spectra were recorded in each condition and processed to evaluate the change in the component band of the spectra corresponding to the amide I (secondary structure) and the CH stretching region (membrane). The IR spectra analysis revealed that expression of PB1-F2 in U937 cells, but not in A549 cells, results in the presence of a specific β-aggregate signature. Furthermore, the lipid membrane composition of U937 cells expressing PB1-F2 was also altered in a cell type-dependent manner. Using DUV microscopy and taking advantage of the high content of tryptophan residues in the sequence of PB1-F2 (5/90 aa), we showed that the increase of the autofluorescent signal recorded in monocytic cells could be correlated with the IR detection of β-aggregates. Altogether, our results constitute an important step forward in the understanding of the cell type-dependent function of PB1-F2., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)