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

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.

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.…”

“…At later stages of an IAV infection PB1-F2 cannot be detected as monomeric within infected cells (Ն8 h.p.i.) but assembled into amyloid oligomers and fibers (22,27,34). In consequence, in the final step of the lytic cycle of influenza virus, PB1-F2 released in extracellular medium is probably assembled into amyloid structures.…”

“…It was previously shown that there is no significant difference in progeny virus titers between wild-type and ⌬F2 viruses upon infection of various cell lines and tissues (22,35). In addition, it was shown that PB1-F2 expression starts at early stages of the viral cycle, when it is barely detectable in its monomeric form (22,27,34). At the later stage of infection the monomeric PB1-F2 is almost undetectable because the protein accumulates as amyloid-like oligomers as has been demonstrated in both A549 and U937 IAV-infected cells (22,27,34).…”

Nanoscale virus biosensors: state of the art

“…The proposed biosensor and specific anti-PB1-F2 monoclonal antibody could be applied for studying PB1-F2 during influenza infection. 122 Miodek et al 123 also described PB1-F2 EB based on immunodetection of the PB1-F2 oligomerization. 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. 123 In the work of Hong et al, 124 concanavalin A was placed on a nanostructured gold electrode, which selectively captured noroviruses. Secondary antibodies conjugated with alkaline phosphatase were used to improve the signal.…”