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 (K_d = 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 (K_off = 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. Nipah virus is a deadly virus that can cause death in up to 70% of infected patients, mostly from fatal inflammation of the brain. Nipah virus is considered a “priority pathogen” for bioterrorism purposes, and it has the potential for widespread economic devastation as it can spread rapidly among susceptible livestock. The authors had previously identified the receptor that mediates Nipah virus entry into cells. This receptor, ephrinB2, is a critical molecule for the development of the vascular and nervous system and is highly expressed on endothelial cells and neurons, which are also the two cell types preferentially infected by Nipah virus in vivo. EphrinB2 belongs to a large family of related molecules that are variably conserved in structure and function. Thus, the authors screened all known ephrins, and found that a closely related molecule, ephrinB3, also can function as an entry receptor for Nipah virus. In addition, the authors established that while ephrinB2 was better used than ephrinB3 as an entry receptor, the same two critical amino acids in ephrinB2 and B3 were responsible for the viral receptor activity of these molecules. The discovery of a more comprehensive set of NiV receptors will aid our understanding of the pathology underlying NiV disease.