Abstract
BACKGROUND: Hendra virus (HeV) is a bat-adapted zoonotic henipavirus belonging to the Paramyxoviridae family. It is classified as a biosafety level 4 (BSL-4) pathogen owing to its broad host range and high fatality rate. Currently, no vaccines or therapeutics are approved for human use. Viral entry is mediated by the attachment (G) and fusion (F) glycoproteins; the heavily glycosylated G protein is responsible for receptor binding. METHODS: The extracellular domain of HeV-G was expressed in Expi293F cells and its glycosylation sites and glycan composition were identified by mass spectrometry. A series of functional assays-including viral entry, receptor binding, cell-cell membrane fusion, antibody neutralization and immunogenicity-were performed to delineate the role of each N-linked glycosylation site. RESULTS: Glycan profiling of HeV-G identified seven N-linked and multiple O-linked glycosylation sites, revealing that the stalk residues (N72, N159) predominantly carry high-mannose glycans, whereas the head-domain N-glycan sites (N306, N378, N417, N481, N529) are primarily modified with complex glycans. Notably, among the head-domain sites, N481 also harbors a substantial proportion of high-mannose glycans. Functional assays revealed that removal of N-glycans at N159, N306 and N417 markedly reduced membrane fusion. The N159 residue is a key site for fusion triggering, and its function is tolerant to specific amino acid substitutions, which may contribute to stabilizing or facilitating the conformational cascade required for F protein-mediated membrane fusion. The N529Q mutant specifically decreased EB3 binding by 2.6-fold, correlating with reduced infectivity. Binding assays with neutralizing antibodies showed that most N-glycan deletions had negligible effects, except that N159Q and N481Q reduced affinity to nAH1.3. Immunization studies in mice demonstrated that N-glycans had minimal impact on humoral immunity, with only minor site-specific differences. CONCLUSION: These findings provide a comprehensive characterization of HeV-G glycosylation, reveal site-specific roles of N-glycans in viral entry, receptor binding and membrane fusion, and offer new insights for vaccine and antibody development.