The detection and evaluation of concentration of influenza virus proteins in biological samples is critical in a broad range of medical and biological investigations regarding the concern over potential outbreaks of virulent influenza strains in animals and humans. This paper describes a sensitive, label-free approach for the detection of a virulence factor PB1-F2. PB1-F2 is a small, 90 amino acid long polypeptide expressed in influenza A viruses, which generally exacerbate virus pathogenicity. The developed immunosensoris based on a non-the-chipcovalently immobilized specific monoclonal anti-PB1-F2 antibody and a SPR technology. The immunosensor was calibrated using purified full length PB1-F2 protein. Itdetected PB1-F2 with the linear range extended from 10 to 500 nM, repeatability of 5% for 500 nM PB1-F2 and showed saturationof protein concentrations higher than 1 μM. The sensor can quantify PB1-F2 in its monomeric form but not when its oligomerization was induced by preincubation in 0.05% SDS. The immunosensor was successfully applied in the detection and quantification of PB1-F2 in infected mouse lungs and cell lines, providing temporal expression profiles of PB1-F2 during viral infection. In lungs of infected mice, the influenza virus structural nucleoprotein NP was detected in parallel using a specific anti-NP antibody. This parallel detection of PB1-F2 and NP suggests that applied sensor chip technology may be amenable to an arrow immunosensor for simultaneous detection of all known influenza virus proteins in infected tissues and cells.

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...

No abstract

It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the αN2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs.ImportanceHuman RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.