Negrete Oscar A, Wolf Mike C, Aguilar Hector C, Enterlein Sven, Wang Wei, Mühlberger Elke, Su Stephen V, Bertolotti-Ciarlet Andrea, Flick Ramon, Lee Benhur
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, California, USA.
PLoS Pathog. 2006 Feb;2(2):e7. doi: 10.1371/journal.ppat.0020007. Epub 2006 Feb 10.
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 (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 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.
埃菲林B2（EphrinB2）最近被发现是尼帕病毒（NiV）的功能性受体，尼帕病毒是一种致命的新兴副粘病毒。埃菲林构成了Eph类受体酪氨酸激酶的一类同源配体，并表现出重叠的表达模式。因此，我们研究了其他埃菲林是否可能作为NiV的替代受体。在此，我们表明，在所有已知的埃菲林（埃菲林A1 - A5和埃菲林B1 - B3）中，除了埃菲林B2外，只有埃菲林B3的可溶性Fc融合蛋白与可溶性NiV附着蛋白G（NiV - G）结合。可溶性NiV - G以亚纳摩尔亲和力（埃菲林B3和B2的解离常数Kd分别为0.58 nM和0.06 nM）与细胞表面的埃菲林B3和B2结合。表面等离子体共振分析表明，NiV - G对埃菲林B3的相对较低亲和力主要是由于更快的解离速率（埃菲林B3和B2的解离速率常数K(off)分别为1.94×10⁻³ s⁻¹和1.06×10⁻⁴ s⁻¹）。埃菲林B3足以允许假型和活NiV的病毒进入。可溶性埃菲林B2和B3能够在表达埃菲林B2和B3的细胞上竞争NiV包膜介导的病毒进入，这表明NiV - G通过重叠位点与埃菲林B2和B3相互作用。突变分析表明，埃菲林B2和B3溶剂暴露的G - H环中的亮氨酸 - 色氨酸残基是NiV结合和进入的关键决定因素。实际上，用亮氨酸 - 色氨酸替换埃菲林B1同源位置的酪氨酸 - 甲硫氨酸残基赋予了埃菲林B1 NiV受体活性。因此，埃菲林B3是NiV进入的真正替代受体，并且埃菲林B类配体G - H环中的两个残基是NiV受体活性的关键决定因素。
