Electrochemical Detection of the Oligomerization of PB1-F2 Influenza A Virus Protein in Infected Cells Anna Miodek, †,‡ Jasmina Vidic,* ,‡ Helene Sauriat-Dorizon, † Charles-Adrien Richard, ‡ Ronan Le Goffic, ‡ Hafsa Korri-Youssoufi,* ,† and Christophe Chevalier* ,‡ † CNRS UMR-8182, Institut de Chimie Molé culaire et de Mate ́ riaux d’Orsay, Equipe de Chimie Bioorganique et Bioinorganique, Bâ timent 420, 91405 Orsay, France ‡ INRA, Unite ́ de Virologie et Immunologie Mole ́ culaires, UR892, Domaine de Vilvert, F-78350 Centre de Jouy-en-Josas, France * S Supporting Information ABSTRACT: PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 μM and from 5 nM to 0.5 μM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent. M any neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease, are characterized by the intracellular or/and extracellular accumulation in tissues and organs of misfolded proteins in the form of fibrillar deposits. Proteins that undergo pathological structural rearrangements to form these fibers are either natively disordered proteins or proteins with destabilized native conformation. The amyloid nature of these assemblies is characterized by a common structural motif of a cross-β-sheet and the same cytotoxic activity associated with neurodegenerative diseases. 1-3 An increasing number of proteins unrelated to amyloid neurodegenerative pathologies are reported to aggregate into amyloid-type assemblies. 4 They switch from a monomeric to an aggregated state that includes amyloid oligomers, prefibrillar assemblies, and mature fibers, sharing the same structural behavior as proteins involved in amyloid diseases. 5 Recently, PB1-F2 protein of influenza A virus (IAV) was shown to form amyloid-like fibers in IAV-infected cells, 6 but the kinetics of fibers formation, their intracellular localization, and their role in the viral cycle remain to be elucidated. Every year, IAV, a member of the Orthomyxoviridae family, 7 spreads on a worldwide scale and affects millions of people. 8 PB1-F2 was discovered in 2001 and was initially described as a proapoptotic protein. 9 Although expression of PB1-F2 is not necessary for developing an influenza infection, PB1-F2 was reported to contribute to the immunopathological disorders developed during infection 10,11 and to enhance secondary bacterial infections. 12 PB1-F2 is a small protein of 87-90 amino acids which displays a strong polymorphism, in sequence and length. 13 PB1- F2 is expressed in most human and avian IAV strains in its full- length version. PB1-F2 was described as a pro-apoptotic factor specifically inducing apoptosis in macrophages and monocytes and, thus, enhancing IAV virulence by compromising viral clearance. 9 In contrast, PB1-F2 has no direct pro-apoptotic effect in epithelial cells. 14 PB1-F2 has a short half-life and is expressed independently of the expression level of other influenza proteins. 9 Although many studies focused on the immunoregulatory function of PB1-F2, very little structural information is available. Previous studies with recombinant PB1-F2 or synthetic peptides have shown that the conformation of PB1- F2 can switch according to the hydrophobicity of its environment. 6,15 PB1-F2 has no secondary structure in aqueous solutions but adopts an α-helical structure within its positively charged C-terminal domain upon addition of trifluoroethanol. 15 Received: May 14, 2014 Accepted: July 22, 2014 Published: July 22, 2014 Article pubs.acs.org/ac © 2014 American Chemical Society 9098 dx.doi.org/10.1021/ac5018056 | Anal. Chem. 2014, 86, 9098-9105