Supplementary Material for: ‘a’-Position-Mutated and G4-Mutated Hemagglutinin-Neuraminidase Proteins of Newcastle Disease Virus Impair Fusion and Hemagglutinin-Neuraminidase-Fusion Interaction by Different Mechanisms Chu F. Wen H. Zhang W. Lin B. Zhang Y. Sun C. Ren G. Song Y. Wang Z. 10.6084/m9.figshare.5123857.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_a_-Position-Mutated_and_G4-Mutated_Hemagglutinin-Neuraminidase_Proteins_of_Newcastle_Disease_Virus_Impair_Fusion_and_Hemagglutinin-Neuraminidase-Fusion_Interaction_by_Different_Mechanisms/5123857 <b><i>Objectives:</i></b> To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. <b><i>Methods:</i></b> We mutated six ‘a’ residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. <b><i>Results:</i></b> Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. <b><i>Conclusions:</i></b> It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures. 2012-10-04 00:00:00 Paramyxovirus Hemagglutinin-neuraminidase protein Heptad repeat region Glycosylation Membrane fusion Hemagglutinin-neuraminidase-fusion protein complexes