Abstract
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.
Highlights
Influenza A viruses (IAVs)2 are responsible each year for seasonal epidemics resulting in considerable illness, death, and important economic losses [1]
We previously showed by thioflavin staining that PB1-F2 knock-out (F2) formed amyloid fibers in vitro using recombinant protein and in IAV-infected cells [25]
Set-up—To determine whether the expression of PB1-F2 drives the formation of amyloid aggregated structures in different IAV susceptible cell types, we infected monocytic cells (U937) and lung epithelial cells (A549) and tried to reveal the presence of such structures using sFTIR and Synchrotron deep ultraviolet (sDUV) microspectroscopy (Fig. 1)
Summary
Influenza A viruses (IAVs) are responsible each year for seasonal epidemics resulting in considerable illness, death, and important economic losses [1]. PB1-F2 was shown to exacerbate the pathogenicity of IAVs in mouse models [11,12,13,14], PB1-F2 attenuates virulence in chicken [15] Such observations are in accordance with the hypothesis that the loss of expression of PB1-F2 in mammals is beneficial for viral fitness, whereas in avian species, PB1-F2 is positively selected to contribute to an optimized spreading of the virus without increased virulence. PB1-F2 was first described as a proapoptotic protein targeting the mitochondria (16 –18) and inducing apoptosis by loss of the mitochondrial membrane potential [19] Another proposed function of PB1-F2 is the modulation of innate immune response [20]. The presence of amyloid fibers in different types of IAV target cells during infection remained to be validated by new techniques
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