Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding.

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic alpha-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.