EphrinB2 was recently identified as a functional cellular receptor for NiV [8
]. EphrinB2 expression on endothelial cells, neurons and smooth muscle cells [10
] is highly consistent with the known tropism of NiV infection [7
]. Here, we show that ephrinB3 is an alternate receptor for NiV and is independently able to support NiV entry and infection, albeit less efficiently than ephrinB2. NiV-G binds to both ephrinB2 and B3 with subnanomolar affinity, with the relatively weaker Kd
of NiV-G for ephrinB3 explained by its faster off-rate. Finally, we implicate two residues (L–W) common in the G–H loop of ephrinB2 and B3 as crucial for NiV receptor activity. Remarkably, replacement of the Y–M residues in the homologous positions in ephrinB1 with L–W conferred wild-type NiV-G binding activity and substantial NiV receptor activity to a protein that is otherwise nonfunctional as a NiV receptor.
To our knowledge, there is no specific indication that ephrinB3 is expressed in the endothelium. At the minimum, ephrinB3 does not appear to be critical to vascular development since ephrinB3 knockout mice lack the overt defects in vascular morphogenesis seen in ephrinB2 knockout mice [23
]. However, NiV entry into microvascular endothelial cells is almost completely abrogated by soluble ephB4-Fc [8
], which binds to ephrinB2 but not B3, suggesting that ephrinB3 is likely not expressed on endothelial cells, at least not at levels that can support robust viral entry. In contrast, ephrinB3 is expressed in the CNS in overlapping and distinct patterns with ephrinB2 [18
]. In the regions of the adult brain such as the cerebral cortex [26
] and the hippocampus [28
] where ephrinB2 and B3 exhibit overlapping expression, NiV could potentially use either receptor for entry with a possible preference for ephrinB2 based on the higher affinity of NiV-G for ephrinB2. However, in regions such as the corpus callosum [27
] and the spinal cord [16
], ephrinB3 is distinctly expressed and could account for specific aspects of NiV pathology.
EphrinB3 knockout mice studies indicate ephrinB3 is expressed in the spinal cord midline and functions to prevent corticospinal tract axons from recrossing the midline. Coincidently, in a histological study of NiV infection by Wong et al., three of eight patients examined showed pathological lesions in the spinal cord similar to other regions of the CNS [7
]. Indeed, clinical symptoms of segmental myoclonus and flaccid tetraplegia combined with nerve conduction studies have also suggested upper cervical and lower spinal cord involvement [29
], and magnetic resonance imaging confirmation of a spinal cord lesion at the cognate C7 level in a patient who developed left-arm dysaesthesia and finger weakness has also been reported [30
]. In another study that reported magnetic resonance imaging findings in eight patients with NiV encephalitis, half the patients had numerous punctate lesions in the corpus callosum [31
], where ephrinB3, but not ephrinB2, is expressed [27
]. Thus, although ephrinB2 seems to be the primary receptor for NiV, ephrinB3 can likely be used as an alternative receptor and may account for some of the CNS pathology seen in NiV infection.
In this study, we also established that NiV-G binding to ephrinB2 and B3 is dependent on the same L–W residues that are important for endogenous ephrinB2/EphB2 interactions [21
]. Since NiV-G interacts with ephrinB2 in a similar fashion with at least some of the Eph B-class receptors, and NiV-G forms higher order oligomers [32
] analogous to Eph B-class receptors [10
], NiV-G could potentially induce “reverse-signaling” upon ephrinB2 or B3 binding.
In vivo, Eph–ephrin interactions cause bidirectional signaling that can direct the migration of endothelial cells and neuronal dendrites [10
]. Therefore, NiV infection may not only target ephrinB2- or B3-expressing cells, but also disrupt normal Eph–ephrin signaling and possibly alter cellular migration patterns. Indeed, infusion of soluble EphB2-Fc has been reported to disrupt the migration of ephrinB2- and B3-expressing cells of the subventricular zone region in an adult mouse [36
]. Although the levels of Ephs and ephrins in other regions of the adult brain are reduced compared to neonatal-stage expression [27
], Eph and ephrins can alter their expression patterns after injury to the spinal cord [37
], hippocampus [38
], or after infection [39
]. In this case, NiV infection may alter ephrin expression patterns in the CNS and disrupt the endogenous Eph–ephrin signaling resulting in the neuropsychiatric [41
] or neuropathologic sequelae seen in NiV infections.
Zoonotic diseases such as those caused by NiV have become an increasing threat in several parts of the world [42
]. The habitat of the pteropid fruit bat, considered as the natural reservoir host, spans from the east coast of Africa across southern and Southeast Asia, east to the Philippines and Pacific islands, and south to Australia [43
]. Although NiV outbreaks have only occurred in Malaysia, Bangladesh, and Singapore, increased surveillance in other geographical regions of the pteropid habitat found bats to harbor NiV [44
]. Therefore, NiV continues to remain a potential threat to both human and animal populations. This underscores the need for the development of antiviral therapeutics. A complete understanding of Nipah viral entry at the level of receptor engagement may help in these efforts.