Using a site-directed mutagenesis strategy, we have identified conformation-dependent receptor binding elements in the predicted globular-head region of the HeV G envelope glycoprotein. The regions identified here as important for receptor engagement consist of residues located in several strands of putative beta sheet 4, as well as two residues residing in beta sheet 1. Our data are complementary to the findings of Guillaume et al. (19
), who identified putative receptor binding residues in NiV G residing in the globular-head region in areas similar to those found to be critical for MeV H receptor binding, although no biochemical or immunological characterizations were made with those mutant glycoproteins. In addition, our data are also consistent with earlier findings of Negrete et al. (32
), where a deletion mutant of NiV G composed of residues 437 to 464 (Δ437-474 NiV G) had lost the ability to bind to the surfaces of receptor-positive 293T cells. According to a proposed structural model of HeV G (38
), residues 437 to 464 lie within β4
, and we have identified five residues important for receptor binding located within this region, namely, G439, K443, G449, K465, and D468, which are crucial to HeV G's ability to bind ephrinB2 or ephrinB3. Thus, we speculate that the binding defect of the Δ437-474 NiV G construct is likely due to the absence of amino acid residues G439, G449, K443, K465, and D468, all of which were found to be important for receptor binding in the present study.
In addition, we found that differences observed in the MAb reactivities of the various G glycoprotein mutants also correlated with their receptor binding phenotypes. Of the six MAbs, three are predicted to bind G at or near the domain(s) of the glycoprotein responsible for receptor binding. Accordingly, it was these three MAbs that showed the most striking differences in the reactivities of the receptor-binding-defective mutants compared to WT G. Specifically, H2.1, 101, and 102.4 showed considerable decreases in their abilities to recognize the mutants G439A, K443A, and K465A, as well as smaller but still noticeable effects on their abilities to recognize D260A and G449A (Table ). The correlation between loss of ephrinB2 and ephrinB3 binding ability and the loss of reactivity to MAbs 101 and 102.4, which were previously shown to block receptor binding (42
), supports our conclusion that the domains of G identified in this study are indeed likely to be involved in receptor binding. In addition, the correlation between losses in receptor binding ability and the decrease in reactivity to H2.1 by these same mutants further supports our hypothesis that HeV G and MeV H may have similarities in the locations of their respective receptor binding sites, as this antibody is thought to bind G in regions analogous to the SLAM-binding domain of MeV H (38
Interestingly, we also found that all of the mutants that were defective in receptor binding were still able to interact with and coprecipitate with their partner glycoprotein, HeV F (Fig. ). Surprisingly, the majority of these mutants that demonstrated decreased receptor binding were found to exhibit an apparent increase in their abilities to coprecipitate with HeV F. This suggests that there is a subtle conformational difference between a mutant G that cannot bind receptor and the WT G. Historically, there have been two competing mechanistic models of paramyxovirus glycoprotein-mediated membrane fusion (reviewed in reference 27
). One model suggests that F and G interact only after receptor binding takes place, and presumably, receptor binding triggers a conformational change in G that facilitates this F interaction. This interaction would be the fusion-promoting activity of G, and subsequently, the F glycoprotein becomes fusion activated, inserts its fusion peptide into target membranes, and facilitates the membrane fusion process. The second model suggests that interaction of the F and G envelope glycoproteins preexists and is independent of any receptor binding event and that it is receptor binding that triggers conformational change in G, which may or may not release F but nevertheless triggers the fusion activity of F. Our present data support this second model, in which F and G interact prior to receptor binding, not only in that WT G coprecipitates with F in the absence of receptor (HeLa-USU cells are receptor negative) (5
), but also in that G glycoprotein mutants that possess significant defects in receptor binding can still coprecipitate with F to levels equivalent to or greater than that of WT G. Although we favor the interpretation that we have removed important residues in G for engaging receptor, an alternative explanation could be that some of these mutant G glycoproteins are adopting a prereceptor-bound conformation that is more favorable for F binding and less favorable for receptor binding.
The effects of several mutations in HeV G on ephrinB2 and ephrinB3 binding did translate into measurable effects on their abilities to promote cell fusion, as predicted. The overall trend observed from the present experiments was that reduced receptor binding capacities of individual G mutants correlated qualitatively with reductions in cell fusion measurements, although we cannot exclude the possibility that the reductions in cell fusion promotion activity observed for some G mutants (D257A, D260A, K443A, G449A, and D468A) could be caused, at least in part, by those mutants' increased abilities to bind HeV F, in addition to their decreased receptor binding phenotypes. Further experiments will be needed to dissect out the contribution of each of these effects to the fusion process. Taken together, the various mutations in HeV G identified here, which impaired its functional activities, appear to be specific for receptor binding and were not due to gross conformational defects, loss of an ability to interact with F, or a lower cell surface expression phenotype.
Notably, in addition to showing that, like NiV G, HeV G could engage ephrinB3 (Fig. ) and use it as a functional receptor in cell fusion (data not shown), we also noted that the same residues within HeV G that appeared critical for binding ephrinB2 were also important for binding ephrinB3. Mutation of residues D257, D260, G439A, K443, G449, K465, and D468 of HeV G to alanine resulted in defects in ephrinB3 binding, similar to the binding pattern observed with ephrinB2. Together with data from Negrete et al. indicating that addition of soluble ephrinB2 can inhibit ephrinB3-dependent entry by NiV and vice versa (32
), the present observations strongly suggest that each G glycoprotein binds to two different members of the ephrin ligand family via a common receptor binding domain. Further study of additional HeV, as well as NiV, G mutations will help to better delineate the domain(s) of G involved in receptor engagement, and it will also be important to confirm these observations in the context of virus infection in future experiments. In summary, our data provide strong biochemical and functional evidence of a conformation-dependent discontinuous ephrinB2 and ephrinB3 binding domain within the henipavirus G glycoprotein and will aid our understanding of the binding and infection processes of these important emerging pathogens.