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

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.