Syncytia generated by HN and F of virulent NDV induce ER stress and UPRmt. A Representative images of F and HN co-operation that increased the interaction between ER and mitochondria in A549 cells. A549 cell were co-transfected with EGFP-Mito and DsRed-ER, together with both Flag-F and HA-HN plasmids, at the indicated time. At 0, 6, 12, 18 and 24 hpt, the coverslips were examined IFA. The plot profile of linear region of interest was analyzed using Image J. B Detection results of ER stress and UPRmt marker proteins in A549 cells through Western blot analysis. C Influence of the cleavage site motif of F on the activation of ER UPR and UPRmt. In A549 cells, wild-type HN plasmid was co-transfected with wild-type F, F112G, F115G, F117L, F112G+115G, F112G+117L, F115G+117L and F112G+115G+117L. At 36 hpt, the Western-blot samples were collected and analyzed. D A model pattern of activated F1 interacts with HN for the disturbance of mitochondrial fusion–fission homeostasis to enhance syncytium formation via the UPR of ER and UPRmt. After HN and F co-transfection, the non-fusogenically F protein precursor (F0) form is proteolytically cleaved into a disulfide-linked F1 + F2 heterodimer, to be fusogenically active, which is an essential for fusion by positioning the hydrophobic fusion peptide (FP) at the newly formed N-terminus of F1 [29, 30]. Then, the fusogenically active F protein and HN form an F + HN complex via the interaction between a stalk and a domain with two heptad repeat regions (HRA and HRB) [29, 30]. Once triggered, HRB completely refolds around HRA, thereby forming six stable HBs and a fusion pore [29, 30]. Apart from spatial distribution rearrangement, the UPR of ER and UPRmt is induced by activated F1 and HN to regulate and disturb mitochondrial fusion–fission homeostasis, which eventually produces hyper-fused and fragmented mitochondria. Eventually, nuclear-aggregated and hyper-fused mitochondria provide additional energy to enhance syncytia formation and membrane fusion.