Two Key Residues in EphrinB3 Are Critical for Its Use as an Alternative Receptor for Nipah Virus

Abstract

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1–A5 and ephrinB1–B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (Koff = 1.94 × 10−3 s−1 versus 1.06 × 10−4 s−1 for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu–Trp residues in the solvent exposed G–H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr–Met residues in the homologous positions in ephrinB1 with Leu–Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G–H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

A broad-spectrum antiviral targeting entry of enveloped viruses

“…Pseudotyped VSV viruses were prepared and assayed for infection as previously described (26,(29)(30)(31). Unless indicated otherwise, all infections were performed in 1% FBS in PBS.…”

“…4 A and B). To see if LJ001-treated virions retain conformationally intact envelope glycoproteins, we performed a virion-cell binding assay by incubating NiV-pVSV viruses, in the presence or absence of LJ001, with CHO cells stably expressing the NiV receptor, ephrinB2 (28)(29)(30) and then assayed for virus binding with antiNiV-F polyclonal antibodies (Fig. 4C) (29,31).…”

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

“…The G protein is embedded in the viral membrane, and its stimulation by host receptors forms the critical first step that facilitates Nipah's entry into host cells. G binds to the ectodomains of specific receptor proteins on the host cell membrane, ephrin B2 and ephrin B3 (8)(9)(10), and this binding stimulates G to activate a second viral membrane protein, F. The activated F protein, in turn, facilitates the fusion of the Nipah and host membranes (Fig. 1).…”

Species-specific and individual differences in Nipah virus replication in porcine and human airway epithelial cells

“…To fulfil this task, the receptor-binding glycoprotein G (NiV G) has to bind to its main host cell receptor, ephrin-B2 (Bonaparte et al, 2005;Negrete et al, 2005). In some neurological tissues, NiV G can additionally use ephrin-B3 as entry receptor, though the binding affinity was shown to be reduced compared to NiV G-ephrin-B2 binding (Negrete et al, 2006). After binding to ephrin, a conformational change in NiV G triggers NiV fusion protein (NiV F)-mediated virus-cell or cell-cell fusion events.…”