Detection of Soluble Oligomers Formed by PB1-F2 Influenza A Virus Protein in vitro

Abstract

PB1-F2 is a short polypeptide of approximately 90 amino acids which is dispensable for the viral replication [1]. PB1-F2 is not encoded by all human and swine IAV strains although nearly all the avian IAV express a full length version of the protein suggesting a host-specific function. It is tempting to correlate the mortality rate of a particular viral strain and the expression of PB1-F2 since contemporary H1N1 strains like the pandemic H1N1/2009 virus that no longer express PB1-F2 appeared to be less virulent. Thus, PB1-F2 is considered as one of the viral protein involved in IAV pathogenicity. PB1-F2 displays a number of original features as a short half-life and rapid proteasomedependent degradation, a mitochondrial tropism but also the ability to localize in the nucleus and cytoplasm of infected cells in a cell-typeand viral strain-dependent fashion [2]. The clear function of PB1-F2 is still unknown even if several groups described apoptotic properties that seem to be influenced by multiple factors such as the primary sequence variability and the range of interactions with other viral proteins and host-specific cellular partners [3]. In a previous study, we showed that PB1-F2 could be classified as a member of the intrinsically disordered proteins due to its lack of structure in aqueous solution and its ability to switch from a random to an alpha-helical or a beta-sheet secondary structure [4]. This conformational change could play an important role in the regulation of PB1-F2 interactions with the host proteins during the viral cycle.There are an increasing number of proteins unrelated to any amyloid pathology that are reported to aggregate in vitro and/or in vivo into polymer assemblies of amyloid type [5]. This feature is probably related to a general tendency of the polypeptide backbone to self-organize into thermodynamically stable polymeric assemblies. Aggregated protein molecules in these fibrillar forms are associated via intra-molecular hydrogen bonds established between the peptide bonds in parallel and anti-parallel beta-strand conformations [6]. In several cases, lipid membranes were shown to favor amyloid fibrillogenesis [5]. We previously shown that negatively charged liposomes in presence of low concentration of recombinant PB1-F2 were destroyed and resulted in amyloid aggregation of PB1-F2 [4]. Furthermore, using Thioflavin S that binds specifically to beta-strand repetitive motives in amyloid fibers, PB1-F2 amyloid structures were detected in membrane vicinities of cells infected with influenza virus [4]. As observed with negatively charged lipids, an anionic detergent, sodium dodecyl sulfate (SDS), in conditions below its critical micellar concentration was shown to induce PB1-F2 aggregation of amyloid fibers [4,7]. In consequence, SDS appears to be a good reagent to trigger and study PB1-F2 oligomerization process in vitro.The putative role of fibrillated influenza PB1-F2 protein in IAVinfected cells is still unknown and new analytical approaches are needed for its elucidation. In this st...

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

“…6, A and B). We previously showed that PB1-F2 converts into amyloid-like structures in the presence of a diluted anionic detergent SDS (concentration of SDS below its CMC of 0.23% (w/v)) (27,29,34). The long PB1-F2 fibrils obtained were of micron sizes and showed no significant cytotoxic effect on A549 cells (Fig.…”

“…Spectroscopic analyses revealed the presence of ␣-helical structures within PB1-F2 only in the concentrated TFE solution, which is far from physiological conditions. In the membrane mimic environment, PB1-F2 was demonstrated to aggregate to amyloid-like structures with a characteristic amyloidal cross-␤-sheet secondary structure (27)(28)(29). Additionally, PB1-F2 can adopt ␤-sheet conformation and oligomerize to amyloid structures within infected cells, as shown for IAV-infected monocytes and epithelial cells (27,28).…”

Electrochemical Detection of the Oligomerization of PB1-F2 Influenza A Virus Protein in Infected Cells

“…XXXX, XXX, XXX−XXX C Biosensing Recombinant PB1-F2 in Monomeric and Oligomeric States. Two antibodies were used to detect PB1-F2: mAb R14, which specifically recognizes the monomeric random coiled form of PB1-F2,6,20 and pAb A11, a conformational antibody raised against a generic conformational β-sheet epitope present in amyloid oligomers 1,22. The elaboration of an…”

N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes

“…Full length PB1-F2(1-90) converts from random into amyloid-like structures in a diluted solution of anionic detergent SDS (concentration of SDS below its CMC of 0.23 %w/v) [13,15,17]. To determine if PB1-F2 (1-52) and PB1-F2 (53-90) also convert into amyloid-like structures, the proteins were dissolved in 10 mM sodium-acetate buffer, pH 5 containing 0.05 %v/w SDS.…”