Influenza virus protein PB1-F2 interacts with CALCOCO2 (NDP52) to modulate innate immune response
Résumé
PB1-F2 is a viral protein encoded by influenza A viruses (IAVs). PB1-F2 is implicated in virulence by triggering immune cell apoptosis and enhancing inflammation. To obtain an insight into the molecular mechanisms of PB1-F2-mediated virulence, we used the yeast two-hybrid approach to find new PB1-F2 cellular interactors. This allowed us to identify calcium-binding and coiled-coil domain 2 (CALCOCO2, also known as NDP52) as a binding partner of PB1-F2. Binding of PB1-F2 to CALCOCO2 was confirmed by pull-down. Surface plasmon resonance binding experiments enabled us to estimate the dissociation constant (Kd) of the two partners to be around 20 nM. Using bioinformatics tools, we designed a CALCOCO2 interaction map based on previous knowledge and showed a strong connection between this protein and the type I interferon production pathways and the I-\kappaB kinase/NF-\kappaB signalling pathway. NF-\kappaB reporter assays in which CALCOCO2, MAVS and PB1-F2 were co-expressed showed a cooperation of these three proteins to increase the inflammatory response. By contrast, PB1-F2 inhibits the TBK1-dependent activation of an ISRE reporter plasmid. We also demonstrated that the signal transducer TRAF6 is implicated in the enhancement of NF-\kappaB activity mediated by PB1-F2/CALCOCO2 binding. Altogether, this report provides evidence of an interaction link between PB1-F2 and human proteins, and allows a better understanding of the involvement of PB1-F2 in the pathologic process mediated by IAV.
Mots clés
- mitochondrial antiviral signalling protein
- MAVS
- qRT-PCR
- quantitative reverse transcription PCR
- SPR
- surface plasmon resonance
- siRNA
- small interfering RNA
- TRAF6
- TNF receptor-associated factor 6
- TRAF6 DN
- dominant-negative form of TRAF6
- TRAF6 FL
- full-length TRAF6
- Y2H
- yeast two-hybrid system
- RU
- resonance unit
- influenza virus
- innate immune response
- Nuclear Proteins
- Protein Binding
- Protein Interaction Mapping
- Surface Plasmon Resonance
- Computational Biology
- Host-Pathogen Interactions
- Humans
- Immunity
- Innate
- Influenza A virus
- Kinetics
- Two-Hybrid System Techniques
- Viral Proteins
- Virulence Factors
- PB1-F2
- CALCOCO2
- calcium-binding and coiled-coil domain 2
- IAV
- influenza A virus
- ISRE
- IFN-stimulated response element