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

Abstract

PB1-F2 is a virulence factor of influenza A virus (IAV) whose functions remain misunderstood. The different roles of PB1-F2may 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 wildtype 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 ␤-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 polymerase 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.e. of 79 amino acids or more (5, 6). Full-length PB1-F2 was shown to contribute to the virulence of IAVs (7), notably of highly virulent A/H5N1 (8) and of the Spanish flu virus of 1918 (9). Fulllength PB1-F2 was also expressed by the pandemic viruses of 1957 and 1968 (6). Nevertheless, the last 2009 pandemic A/H1N1 virus expressed only an 11-amino acid C-terminaltruncated protein as a result of the accumulation of premature stop codon (10). Although PB1-F2 was shown to exacerbate the pathogenicity of IAVs in mouse ...

The Influenza Virus Protein PB1-F2 Increases Viral Pathogenesis through Neutrophil Recruitment and NK Cells Inhibition

“…The wt virus infection is also associated with dysregulation of PB1-F2 Impacts Immune Cell Recruitment genes implicated in "sensory perception of chemical stimulus" and in "ion transport"; the first group of 23 genes is mainly composed of olfactory receptor genes and the second group of genes is composed of transporter and ion channels. These functional responses specifically associated to the PB1-F2 expressing virus are likely representative of a deleterious effect on the epithelium that could be mediated by the intrinsic ability of PB1-F2 to alter membrane properties [27,31,37,38]. These gene expression profiles could therefore reflect the tissue remodeling processes that resulted from the damage done to the respiratory epithelium.…”

“…On the virus side, the PB1-F2 protein has also been shown to increase the pathology by exacerbating leukocyte recruitment within the airways [24,25]. Expression of this protein by the IAV-infected cells can induce membrane disruption and cell death through amyloid oligomers formation [26,27]; also, PB1-F2 expression increases the NF-kB activity within the infected cells [28]. This protein is therefore considered as an IAV virulence factor.…”

Influenza A virus PB1-F2 protein: An ambivalent innate immune modulator and virulence factor

“…Indeed, structural analysis reveals that PB1‐F2 forms amyloid β aggregate in membranous environment 44,45 . PB1‐F2 protein changes from monomer to higher‐order oligomer or protein aggregate during lytic IAV life cycle, more rapidly in U937 monocytic cell line than A549 lung cell line 46,47 . In line with this, PB1‐F2‐mediated apoptotic response is more pronounced in immune cells, such as monocytes and macrophages 17,32,48,49 .…”

“…Moreover, PB1‐F2 protein can bind to the transmembrane domain of MAVS 74 . As PB1‐F2 is a self‐aggregating protein that forms amyloid structure as mentioned earlier, 44‐47 PB1‐F2‐bound MAVS protein is probably sequestered and inactivated. Importantly, it was demonstrated that N66S mutation of PB1‐F2 substantiates its suppressive effect on RIG‐I‐dependent type I IFN production at the level of MAVS, mediated through higher binding affinity to MAVS and more pronounced dissipation of ΔΨm 74,75 .…”

Virulent PB1-F2 residues: effects on fitness of H1N1 influenza A virus in mice and changes during evolution of human influenza A viruses

“…PB1-F2 can increase cell death, potentiate inflammatory responses, impair cellular innate immunity, and upregulate viral polymerase activity 16 , 18 – 35 . Naturally-occurring variations in PB1-F2 amino acid sequence have been linked to its virulence 15 .…”