Here we show that both ephrinB2 and -B3 can serve as functional receptors for NiV and HeV. While NiV and HeV can use ephrinB2 equivalently well, we initially found that HeV (i.e., H-507S) uses ephrinB3 much less efficiently than does NiV. Mapping studies indicated that the valine 507 residue in NiV-G can confer efficient ephrinB3 usage when placed in the context of HeV-G. Upon careful inspection, we found that two alternative published HeV-G sequences exist, differing by one amino acid at residue 507 in the globular domain (23
) (GenBank accession no. AF01749.2 and AF01749). Further analysis indicated that the HeV sequence with a threonine at residue 507 (H-507T) in the attachment protein has comparable ephrinB2 and -B3 usage to that of NiV, while a serine at residue 507 can significantly reduce HeV and NiV usage of ephrinB3 without compromising their ability to use ephrinB2 efficiently.
The frequency at which these two alternative HeV-G sequences exist in natural viral isolates is currently unknown. Perhaps these particular viral isolates reported in GenBank were sequenced from a mixed population of viruses. RNA viruses are prone to high mutation rates due to the lack of proofreading ability of their RNA-dependent RNA polymerases. Thus, RNA viruses exist as quasispecies, which can be an intrinsic property of the virus that determines its pathogenicity (44
). In order to determine which HeV-G sequence is the more prominent “wild-type” sequence, more HeV isolates need to be sequenced directly from infected sources or from early-passage stocks. Indeed, multiple horse, bat, and human isolates appear to have been reported (15
), although their sequences have not been made public.
With regards to NiV, only minor changes have been reported between pig, bat, and human NiV isolates from the Malaysian outbreak, but more dramatic changes were seen in viral isolates from the Malaysian and Bangladeshi outbreaks (1
). Since our results indicate that even a slight conservative change in the putative receptor binding domain, such as that between serine and threonine, can significantly affect receptor usage, it would be interesting to determine if the Malaysian, Bangladeshi, and various animal NiV isolates differ in their efficiencies of ephrinB2 versus -B3 usage. Strikingly, a serine-to-threonine change in the receptor binding site of the severe acute respiratory syndrome (SARS) coronavirus spike protein has also been implicated in its ability to bind efficiently to the human ACE2 receptor (30
). Indeed, structural evidence indicates that the terminal methyl group of the critical threonine fills a hydrophobic cavity in the ACE2 receptor which stabilizes the envelope-receptor interaction (29
). As with the SARS viral isolates, we hypothesize that a serine-to-threonine change in the receptor binding region of the henipavirus attachment protein can alter the pathogenicity of the virus via its ability to affect ephrinB3 usage (see below and Fig. ).
FIG. 8. EphrinB2 and -B3 are coexpressed in the cerebral cortex, but ephrinB3 is distinctly expressed in the brain stem. (A and B) A three-dimensional model of the adult mouse brain was colored differently to represent the three major brain regions, i.e., the (more ...)
Guillaume et al. (20
) described a reduction in cell-cell membrane fusion in Vero cells and ephrinB2-transfected CHO cells when they explored some of the same NiV-G mutants that we used. They did not explore our position 507 mutants, but the proximity of residue 507 to the putative receptor binding site identified by their group prompted us to carefully examine their mutations in the context of ephrinB2 and -B3 usage. We found that residues 504 and 505, in addition to residue 507, reduce ephrinB3 receptor usage without compromising ephrinB2 usage. Also, the E533Q mutation was the most potent fusion-nullifying mutation in both of our studies, although we now attribute that phenotype to a lack of both ephrinB2 and -B3 binding. Therefore, we confirm and extend the findings of Guillaume et al. (20
) by attributing the underlying phenotype of their fusogenic mutants to a specific defect in ephrinB2 and/or ephrinB3 receptor usage. In addition, a recent study by Bishop et al. (4
) also identified residues important for henipavirus receptor binding. These residues (G439, K443, G449, K465, and D468) are clustered in beta-sheet 4 strands 1 to 3 (β4S1-3), which are actually not predicted to be on the surface of the globular domain by either their own modeled structure (46
) or the one published by Guillaume et al. (20
). The authors themselves raised the possibility that the identified residues form a discontinuous epitope that affects receptor binding via indirect means.
We previously characterized two overlapping residues (Leu-Trp) at the tip of the solvent-exposed G-H loop of ephrinB2 and -B3 that are critical determinants of NiV binding and entry (38
). The cocrystal structure of ephrinB2 in complex with EphB4 (an endogenous ephrinB2 receptor) (11
) indicates that the G-H loop of ephrinB2 inserts into a hydrophobic canyon of EphB4 (Fig. ). On closer inspection, several residues in the G-H loop of ephrinB2, on either side of the L-W tip, also make substantial contacts with the binding cleft of EphB4 (Fig. ). Since the G-H loop is an important region of contact with EphB4, EphB2 (24
), and NiV-G, we hypothesize that residues in the G-H loop that are different between ephrinB2 and -B3 may interact with the distinct ephrinB3 binding determinants on NiV-G. Therefore, to correlate our previous findings (38
) with current data, we propose that the tip of the G-H loop containing the L-W residues in ephrinB2 and -B3 interacts with residues at or around position 533 in NiV-G, since a mutation at this residue affected both ephrinB2 and -B3 binding. As a consequence, the differing residues in ephrinB2 and -B3 immediately surrounding the L-W tip of the G-H loop (Fig. ) are positioned to interact with distinct ephrinB3 binding determinants (residues 504, 505, and 507) in NiV-G. Interestingly, when the putative ephrinB2 and -B3 binding determinants were mapped onto a published model of NiV-G (20
), we noted that the receptor binding face of NiV-G also has a cleft or canyon, where residue 533 is positioned at one end of the canyon and residues 504, 505, and 507 line the “walls” of the canyon, not unlike the ephrinB2-interacting canyon seen in EphB4.
FIG. 7. NiV-G model and proposed site of ephrinB2 and -B3 interaction in comparison to the crystal structure of the ephrinB2-EphB4 complex. (A) The crystal structure of an ephrinB2 monomer (blue cartoon representation) in complex with its cognate receptor, EphB4 (more ...)
Finally, we suggest that ephrinB3-mediated entry may actually be the ultimate cause of death in acute Nipah viral encephalitis. High-resolution expression mapping data using the Allen Brain Atlas (28
) indicate that while ephrinB2 and -B3 are coexpressed in overlapping regions of the mouse cerebrum (Fig. ; compare panels C and E), ephrinB3, but not ephrinB2, is expressed in the brain stem (Fig. ; compare panels D and F). Tissue gene expression studies with humans have also found that ephrinB3, but not ephrinB2, is expressed in the pons region of the brain stem (43
). These findings underscore the biological relevance of ephrinB3-mediated usage, as severe brain stem dysfunction is the major clinical defining feature of fatal Nipah viral encephalitis (19
). In addition, histological data from fatal cases of NiV infection found abundant NiV antigen staining in the pons region of the brain stem (48
), while magnetic resonance images of NiV-infected patients also found lesions in the pons in three of eight persons examined (33
). Thus, the ability of NiV or HeV to use ephrinB3 efficiently may be a key pathogenic determinant in Nipah viral encephalitis. Indeed, we hypothesize that mutations that enhance ephrinB3 usage may result in a more encephalopathic virus.
More data are required in several henipavirus research areas to determine if alterations in receptor usage by NiV or HeV may account for differences seen in transmission efficiency to humans, tropism, and/or severity of the disease they cause. For example, it was noted that NiV caused more severe infection in the respiratory epithelium than did HeV in a feline model of henipavirus infection, suggesting a concrete example to examine if differential receptor usage contributes to the variant pathological profiles observed (25
). In addition, the use of a recently described NiV rescue system (50
) to generate a virus that can use ephrinB2, but not ephrinB3, to infect an animal model that recapitulates NiV neuronal disease may help to dissect the role of ephrinB3 in fatal Nipah viral encephalitis. A more comprehensive delineation of henipavirus receptor interactions will thus aid our understanding of the pathology underlying this emergent infectious disease.