Identification of Hendra Virus G Glycoprotein Residues That Are Critical for Receptor Binding

Abstract

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformationdependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

Probing the Spatial Organization of Measles Virus Fusion Complexes

“…The general strategy of coprecipitating one envelope glycoprotein with antibodies directed against the other after lysis of the producer cells has been applied to multiple members of the paramyxovirus family (2,5,19,44,75). However, in this setup, the possibility that removal of the stabilizing lipid bilayer induces conformational changes in the metastable prefusion F trimer (76) prior to co-IP, potentially distorting results, cannot be excluded.…”

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

“…10A, three (NiV G 181A , NiV G 2A , and NiV G 6A ) showed 70 to 80% expression compared to wt NiV G. Only NiV G 182A had a significantly lower expression (35% of wt NiV G). The receptor binding capacity of the mutated NiV G proteins was evaluated by quantification of binding to soluble ephrin B2 since ephrin B2 (but not ephrin B1) acts as a cellular receptor for NiV (45)(46)(47)(48)(49)(50)(51). Cells expressing the indicated proteins were allowed to bind ephrin at 4°C for 60 min, and bound ephrin was quantified by FACS.…”

Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements

“…8 b also shows the conformational ensembles of selected residues, including those near the RBD-FAD interface. Experiments show that the alanine-substitution of D468 impacts G-stimulation negatively, indicating that it may be part of the signal transduction pathway (57). We note, however, that in both the wild-type and mutant forms, ephrin induces a negligible shift in D468's conformational ensemble, with h ¼ 0:52, h m ¼ 0:53 (see also Fig.…”