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Synchrotron Infrared and Deep UV Fluorescent Microspectroscopy Study of PB1-F2 β-Aggregated Structures in Influenza A Virus-infected Cells
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Edité par CCSD ; American Society for Biochemistry and Molecular Biology -
International audience. 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) micros-copies at the single-cell level. Cells were infected with a wild-type IAV and its PB1-F2 knockout 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-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-aggregates. Altogether, our results constitute an important step forward in the understanding of the cell type-dependent function of PB1-F2. Influenza A viruses (IAVs) 2 are responsible each year for seasonal epidemics resulting in considerable illness, death, and important economic losses (1). IAVs are also major pathogens of animals and cause devastating outbreaks in domestic poultry and massive culling to control the viral spread. IAVs are members of the Orthomyxoviridae family and their genome is constituted of eight segments of single-stranded negative sense RNA (2). The viral segment 2 encodes the polymer-ase subunit PB1 and two additional proteins, N40, a N-truncated version of PB1 devoid of transcriptase activity, and PB1-F2 from an alternative reading frame (3). PB1-F2 is a small protein of 90 amino acids with a strong polymorphism in sequence and length depending on the viral strain. Although PB1-F2 is expressed in its full-length version in 96% of A/H5N1 avian strains (4), less than 10% of A/H1N1 human viruses isolated since 1949 express a functional version of PB1-F2, i.
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