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Published on: November 2, 2018
The influenza virus protein PB1-F2 interacts with IKKβ and modulates NF-κB signalling
Ana Luísa Reis1, John W McCauley
1Division of Virology, MRC National Institute for Medical Research, London, United Kingdom.
Abstract:
PB1-F2, a protein encoded by a second open reading frame of the influenza virus RNA segment 2, has emerged as a modulator of lung inflammatory responses but the molecular mechanisms underlying this are only poorly understood. Here we show that PB1-F2 inhibits the activation of NF-κB dependent signalling pathways in luciferase reporter assays. PB1-F2 proteins from four different viruses interact with IKKβ in yeast two-hybrid assays and by co-immunoprecipitation. PB1-F2 expression did not inhibit IKKβ kinase activity or NF-κB translocation into the nucleus, but NF-κB binding to DNA was severely impaired in PB1-F2 transfected cells as assessed by Electrophoretic Mobility Shift Assay. Neither the N-terminal 57 amino acid truncated forms nor the C-terminus of PB1-F2 were able to inhibit NF-κB dependent signalling, indicating that the full length protein is necessary for the inhibition.
Insights
Influenza PB1-F2 protein inhibits nuclear factor-kappa B (NF-κB) signaling by impairing DNA binding, not translocation. Full-length PB1-F2 is essential for this modulation of lung inflammation.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Influenza virus PB1-F2 protein modulates lung inflammatory responses.
- The precise molecular mechanisms of PB1-F2's immunomodulatory effects remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms by which PB1-F2 influences NF-κB signaling pathways.
- To investigate the interaction of PB1-F2 with key components of the NF-κB pathway.
Main Methods:
- Luciferase reporter assays to assess NF-κB activation.
- Yeast two-hybrid and co-immunoprecipitation assays to study protein interactions.
- Electrophoretic Mobility Shift Assay (EMSA) to evaluate NF-κB DNA binding.
Main Results:
- PB1-F2 was shown to inhibit NF-κB dependent signaling pathways.
- PB1-F2 proteins from four influenza strains interacted with IKKβ.
- NF-κB translocation was not inhibited, but DNA binding was significantly impaired by PB1-F2.
- Truncated forms of PB1-F2 lacking either the N-terminal 57 amino acids or the C-terminus did not inhibit NF-κB signaling.
Conclusions:
- Full-length PB1-F2 protein is required to inhibit NF-κB dependent signaling.
- PB1-F2 inhibits NF-κB activation by preventing DNA binding, rather than affecting IKKβ kinase activity or nuclear translocation.
- These findings reveal a novel mechanism for influenza virus modulation of host inflammatory responses.
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