Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demon-strate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) andmeasles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDVHN andMVH that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven intro-duced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the abil-ity of the G protein to complement the homologous F protein in the promotion of fusion. Themostmembrane-proximal addedN-glycan reduces fusion by 80%.However, unlike similarNDVHNandMVHmutants, theNiVGglycosylation stalkmutants retain the ability to bind F, indicating that the fusion deficiency of thesemutants is not due to prevention of theG-F interaction. These finding