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RIG-I and MDA5 are two key pattern recognition receptors that sense the invasion of RNA viruses and initiate type I interferon (IFN) response. Although these receptors are generally conserved in vertebrates, RIG-I is absent in chickens, whereas MDA5 is present. Chicken MDA5 (chMDA5) plays a pivotal role in sensing the invasion of RNA viruses into cells. However, unlike mammalian MDA5, where there are in-depth and extensive studies, regulation of the chMDA5-mediated signaling pathway remains unexplored. In this study, we performed a pulldown assay and mass spectrometry analysis to identify chicken proteins that could interact with the N terminal of chMDA5 (chMDA5-N) that contained two CARDs responsible for binding of the well-known downstream adaptor MAVS. We found that 337 host proteins could potentially interact with chMDA5-N, which were integrated to build a chMDA5-N–host association network and analyzed by KEGG pathway and Gene Ontology annotation. Results of our analysis revealed that diverse cellular processes, such as RNA binding and transport and protein translation, ribosome, chaperones, and proteasomes are critical cellular factors regulating the chMDA5-mediated signaling pathway. We cloned 64 chicken genes to investigate their effects on chMDA5-mediated chicken IFN-β production and confirmed the association of chicken DDX5, HSPA8, HSP79, IFIT5, PRDX1, and hnRNPH2 with chMDA5-N. In particular, we found that chicken hnRNPH2 impairs the association between chMDA5-N and MAVS and thus acts as a check on the chMDA5-mediated signaling pathway. To our knowledge, this study is the first to analyze the chicken MDA5–host interactome, which provides fundamental but significant insights to further explore the mechanism of chicken MDA5 signaling regulation in detail.

hnRNPH2 as an Inhibitor of Chicken MDA5-Mediated Type I Interferon Response: Analysis Using Chicken MDA5–Host Interactome