Abstract
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 (beta6S4/beta1H1). 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.
Highlights
Past studies have identified regions in either the fusion (F) or attachment (G/H/HN) glycoproteins that are important for membrane fusion or F-G/H/HN association [2,3,4,5,6,7,8,9,10], the region(s) in G important for receptor-activated triggering of F-mediated fusion remains unknown
Enhanced Binding of Monoclonal Antibody 45 (Mab45) to Nipah virus (NiV)-G (G) Induced by EphrinB2 Receptor Binding—Receptorinduced conformational epitopes can be suggestive of conformational changes in viral envelopes that eventually lead to membrane fusion and viral entry
Binding to coreceptor after CD4-induced conformational changes in HIV gp120 triggers fusion peptide exposure in gp41 and the subsequent six-helix bundle formation that eventually leads to membrane fusion [23, 24]
Summary
NiV, Nipah virus; G/H/HN, attachment glycoprotein; F, and fusion glycoprotein; RBE, receptor binding-enhanced; CD, circular Dichroism; HR, heptad repeat region; MFI, mean fluorescence intensity; HA, hemagglutinin; CHO, Chinese hamster ovary; WT, wild type; Mab, monoclonal antibody 45; FP, fusion peptide. For Paramyxovirus F proteins, the C-terminal HR2 region is generally thought to be pre-formed, but the N-terminal HR1 region is formed only upon F-triggering and FP insertion [11, 13] The formation of this trimeric HR1 core just before six-helix bundle formation, is known as the pre-hairpin intermediate. Despite their common features, viral fusion proteins vary in their detailed structures, triggering factors, and number of viral surface proteins involved. We analyze the early steps in the fusion cascade for NiV and identify a specific region in NiV-G distinct from the receptor binding site that is involved in 1) B2-induced changes that trigger FP exposure in F, 2) modulating the avidity of F/G interactions resulting in displacement of F from G, and 3) transducing receptor-induced membrane fusion. Our results offer testable hypotheses as to whether this model of fusion cascade holds true for other paramyxoviruses that use protein-based receptors
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