A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the Fusion Glycoprotein (F)

Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation sit...

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Bibliographic Details
Published in:	The Journal of biological chemistry 2009-01, Vol.284 (3), p.1628-1635
Main Authors:	Aguilar, Hector C., Ataman, Zeynep Akyol, Aspericueta, Vanessa, Fang, Angela Q., Stroud, Matthew, Negrete, Oscar A., Kammerer, Richard A., Lee, Benhur
Format:	Article
Language:	eng
Subjects:	Animals Antibodies, Monoclonal - pharmacology Chlorocebus aethiops CHO Cells Cricetinae Cricetulus Ephrin-B2 - genetics Ephrin-B2 - metabolism Epitopes - metabolism Humans Mutation Nipah Virus - genetics Nipah Virus - metabolism Peptide Mapping - methods Protein Structure, Tertiary - physiology Vero Cells Viral Envelope Proteins - antagonists & inhibitors Viral Envelope Proteins - genetics Viral Envelope Proteins - metabolism Virus Internalization - drug effects
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Summary:	Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (β6S4/β1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.
ISSN:	0021-9258 1083-351X