A key region of molecular specificity orchestrates unique ephrin-B1 utilization by Cedar virus

An expanded hydrophobic cavity within the structurally constrained receptor-binding site of the Cedar virus attachment glycoprotein facilitates idiosyncratic utilization of ephrin-B1.

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Overall, the paper is written clearly, and the findings represent an important advancement for HNV viral entry. However, the major concern is the novelty. There is a paper published on PNAS recently, titled "Structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by Cedar virus". They demonstrate that CedV can use ephrin-B1, A2, A5 to enter cells, and determined the CedV-G structure at 3.7-Å resolution, and the complex structures of CedV-G with ephrin-B1 or B2 at 3.5-Å and 2.85-Å respectively. It seems this PNAS paper has more data, especially they have the exact affinity data of the protein binding . Both two studies have similar conclusions. Minor concerns: In Figure 6, it is better to add a panel showing the sequence alignment of different HNV-G, to highlight the critical binding sites.
Reviewer #2 (Comments to the Authors (Required)): In this manuscript, Pryce et al demonstrate that EphrinB1, in addition to EphrinB2, but not EphrinB3, is a functional receptor for CedV -a member of the Henipavirus (HNV) genus. The data are supported by comprehensive structural/biochemical experiments including binding assay, pseudovirus entry assay and structural analysis. Utilization of EphrinB1 as an entry receptor has not been reported for any members of the HNV genus before this paper and a recently published paper by Laing et al (PMID: 31548390). This is an important study as it reveals the ability of HNVs to use different members of the Ephrin protein family for entry and shed light on the molecular barriers that dictate specific receptor usage by different HNVs. The data are well presented and the discussion section is thorough. This reviewer has several comments/suggestions described below: In the 2nd part of the Results section, since the authors use ephrin constructs that are fused to Fc, their dimeric nature leads to avidity and artificially enhance the affinity. Thus, these are not genuine Kds.
The authors should cite and discuss the recently reported findings by Liang et al (PMID: 31548390) (including comparing their structure to theirs).
Reported buried surface area (BSA): the authors should clarify that the values in this manuscript are total BSA on EphrinB1 and CedV G (to be consistent with Liang et al, which reported the values on one interface only).
The authors state "Together, binding-induced structural transitions within both CedV-G and the ephrin G-H loops support a model of an induced-fit mechanism of ephrin recognition that is conserved across ephrin-tropic HNVs ".
How did the authors distinguish between true induced-fit and selection from a conformational equilibrium?
"Similarly, both ephrin-B2-Fc and ephrin-B1-Fc inhibited CedVpp entry into CHO-B2 cells (Fig. 5d, right panel), evidencing the ability of ephrin-B1 to block ephrin-B2-dependent CedVpp entry through competition for an overlapping binding site on CedV-G. Moreover, ephrin-B2-Fc inhibited CedVpp entry into CHO-B1 cells (Fig. 5d, left panel). In both CHO-B2 and CHO-B1 cells, ephrin-B2-Fc-mediated inhibition of CedV-G was more potent than ephrin-B1-Fc (Fig. 5d), further supporting our binding (Fig. 2) and entry (Fig. 3) data that suggest ephrin-B2 is more efficiently utilized than ephrin-B1. " Are the structural data consistent with the authors' claim that CedV G utilize EphrinB2 more efficiently than it does EphrinB1? Or what could rationalize this preferred utilization? Also, these results confirm the recently reported structures by Laing et al (PMID 31548390) and this should be mentioned.
How do the authors reconcile the virtually identical binding responses of NiV G to ephrin-B2 or ephrin-B3 (Fig 2 a) with the 3 log difference observed between ephrin-B2 or ephrin-B3 for inhibition of NiVpp entry in CHO-B2 cells (Fig 5 d)?
"Although CedV-G is unable to utilize ephrin-B3, our structural hypothesis suggests that acquired ephrin-B1 specificity does not necessarily come at the expense of ephrin-B3 usage, as the LW motif is common to both ephrin-B2 and ephrin-B3." Could the authors use their structural data to explain why Ephrin B3 is incompatible with CedV G?
Could the authors discuss how conserved EphrinB1 is among different species and whether CedV can transmit among species by utilizing EphrinB1?

The Editorial Board Life Science Alliance
Dear Dr. Leibfried, We wish to submit the revised manuscript by Pryce and Azarm et al. entitled 'A key region of molecular specificity orchestrates unique ephrin-B1 utilization by Cedar virus' to be considered for publication in Life Science Alliance. We thank the reviewers for their responses, and below we address the comments made point by point (our responses in blue and changes to the text in red).

Reviewer #1:
In the manuscript, Rhys et al. report CedV could use not only ephrin-B2, which is a common HNV receptor, but also ephrin-B1 as an entry receptor. They determined the crystal structure of CedV-G protein at a resolution of 2.78-Å, and the CedV -G bound to ephrin-B1 complex structure at a resolution of 4.07-Å. Structural analyses reveal that diverse HNV-G proteins bind to their distinct ephrin receptors in a conserved binding mode, while subtle structural features of CedV contribute to its unique ephrin ligand specificity.
Overall, the paper is written clearly, and the findings represent an important advancement for HNV viral entry. However, the major concern is the novelty. There is a paper published on PNAS recently, titled "Structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by Cedar virus". They demonstrate that CedV can use ephrin-B1, A2, A5 to enter cells, and determined the CedV-G structure at 3.7-Å resolution, and the complex structures of CedV-G with ephrin-B1 or B2 at 3.5-Å and 2.85-Å respectively. It seems this PNAS paper has more data, especially they have the exact affinity data of the protein binding. Both two studies have similar conclusions.

Response:
We thank the Reviewer for their efficient capsulation of our study. This manuscript, in its exact form, was deposited into bioRxiv before the PNAS study was published, or even available online. This journal has editorial policies that do not make prior publications a consideration in evaluating manuscripts under their scoop protection policies. We believe in the open and transparent peer review system that Life Sciences Alliance follow. We agree with the reviewer that any comparisons with the PNAS study by Laing et al. should and can be made in open peer review forums.
Minor concerns: In Figure 6, it is better to add a panel showing the sequence alignment of different HNV-G, to highlight the critical binding sites.

Response:
We completely agree with the reviewer that providing the annotated sequence alignment of HNV-G proteins will help add clarity and give additional context to Fig. 6. Indeed, such an annotated sequence alignment with additional structural annotations was provided in Supplementary Figure 1. We felt these data were best presented as an independent figure, given (1) the discontinuous nature of the ephrin-binding site, (2) the size of the resulting sequence alignment, and (3) the additional information content provided by the structural annotations (e.g. contact residues, disulphide bonds, and N-linked glycan sites etc.)

Reviewer #2:
In this manuscript, Pryce et al demonstrate that EphrinB1, in addition to EphrinB2, but not EphrinB3, is a functional receptor for CedV -a member of the Henipavirus (HNV) genus. The data are supported by comprehensive structural/biochemical experiments including binding assay, pseudovirus entry assay and structural analysis. Utilization of EphrinB1 as an entry receptor has not been reported for any members of the HNV genus before this paper and a recently published paper by Laing et al (PMID: 31548390). This is an important study as it reveals the ability of HNVs to use different members of the Ephrin protein family for entry and shed light on the molecular barriers that dictate specific receptor usage by different HNVs. The data are well presented and the discussion section is thorough. This Reviewer has several comments/suggestions described below: In the 2nd part of the Results section, since the authors use ephrin constructs that are fused to Fc, their dimeric nature leads to avidity and artificially enhance the affinity. Thus, these are not genuine K d s.

Response:
We thank the Reviewer for noting this point and allowing us to clarify the text. We agree that avidity effects resulting from Fc-tagged HNV-G proteins may yield avidity enhanced binding and thus different estimates of K d relative to experiments utilizing monomeric proteins. As such, we do not undertake direct quantitative comparison with other studies quoting bimolecular interaction kinetic values. We instead present values for all ephrin-tropic HNV-Gs, to permit quantitative comparison of all HNV-G ephrin pairs utilizing a uniform experimental set-up. To avoid any confusion, we have updated text in this section: Line 159: The section title has been updated to remove the phrase 'nanomolar affinity' and now reads 'CedV-G binds both ephrin-B1 and ephrin-B2'.
The authors should cite and discuss the recently reported findings by Laing et al (PMID: 31548390) (including comparing their structure to theirs).
Response: Since this manuscript was deposited into BioRxiv before the Laing et al. study was published, even online, we were not able to make any reference to it. In line with editorial advice, we now cite the Laing et al. (PMID: 31548390) reference as an endnote following the Acknowledgements section.
The authors state "Together, binding-induced structural transitions within both CedV-G and the ephrin G-H loops support a model of an induced-fit mechanism of ephrin recognition that is conserved across ephrin-tropic HNVs ".
How did the authors distinguish between true induced-fit and selection from a conformational equilibrium?

Response:
We thank the Reviewer for bringing up this important point and agree the presented structures do not provide grounds to distinguish induced fit from conformational selection. As such, we have altered the text as follows: Line 314-316: Differences between the structural states of both CedV-G and ephrin-B1 may represent an induced-fit mechanism of ephrin recognition, which has been postulated for other ephrin-tropic HNV-Gs [36, 37, 45], or selection from a conformational equilibrium.
Furthermore, wording elsewhere has been altered for the purpose of clarity: Line 294-295: Structural plasticity within this region is observed in other HNV-G proteins and their ephrin-bound complexes [36,37].
"Similarly, both ephrin-B2-Fc and ephrin-B1-Fc inhibited CedVpp entry into CHO-B2 cells (Fig. 5d, right panel), evidencing the ability of ephrin-B1 to block ephrin-B2-dependent CedVpp entry through competition for an overlapping binding site on CedV-G. Moreover, ephrin-B2-Fc inhibited CedVpp entry into CHO-B1 cells (Fig. 5d, left panel). In both CHO-B2 and CHO-B1 cells, ephrin-B2-Fc-mediated inhibition of CedV-G was more potent than ephrin-B1-Fc (Fig. 5d), further supporting our binding (Fig.  2) and entry (Fig. 3) data that suggest ephrin-B2 is more efficiently utilized than ephrin-B1. " Are the structural data consistent with the authors' claim that CedV G utilize EphrinB2 more efficiently than it does EphrinB1? Or what could rationalize this preferred utilization? Also, these results confirm the recently reported structures by Laing et al (PMID 31548390) and this should be mentioned.
How do the authors reconcile the virtually identical binding responses of NiV G to ephrin-B2 or ephrin-B3 (Fig 2 a) with the 3 log difference observed between ephrin-B2 or ephrin-B3 for inhibition of NiVpp entry in CHO-B2 cells (Fig 5 d)?
Response: We thank the reviewer for noting this 2-log (not 3-log) difference. Indeed, we also detect a similar difference in the ephrin-B2 and ephrin-B3 IC 50 values of NiVpp entry inhibition on the Vero-CCL81s (Figure 2b). We have previously noted this apparent discrepancy (e.g. Ref 46: Negrete, O.A. et al., PLoS Pathog, 2006;compare Figures 1B & 3), which is a known observation that has repeatedly appeared in the literature. Envelope-receptor interactions that lead to binding versus inhibition of entry measure two different things. Binding measures direct protein-protein interactions under equilibrium conditions, whereas entry involves a host of allosteric signals from receptor binding, to F-triggering, to fusion-pore formation, which then results in delivery of the viral RNP into the host cell cytoplasm, and subsequent transcription and viral genome replication that leads to detection of the reporter gene signal. Thus, small differences in binding affinity, and more importantly, differences in the efficiency of how particular receptors (e.g. ephrin-B2 versus ephrin-B3) allosterically trigger F proteins (not measured by receptor binding affinities alone) can amplify any putative differences in envelop-receptor interactions that result in entry. How different receptors mechanistically trigger productive fusion and entry by various paramyxoviruses is a subject of intense investigation by aficionados of paramyxovirus entry and is beyond the scope of this current study.
"Although CedV-G is unable to utilize ephrin-B3, our structural hypothesis suggests that acquired ephrin-B1 specificity does not necessarily come at the expense of ephrin-B3 usage, as the LW motif is common to both ephrin-B2 and ephrin-B3." Could the authors use their structural data to explain why Ephrin B3 is incompatible with CedV G?

Response:
We thank the Reviewer for the opportunity to clarify this point. The precise structural determinants that prevent recognition of ephrin-B3 by CedV-G are presently unclear. Indeed, answering such questions is a focus of ongoing work. We have updated the Discussion to highlight this uncertainty rather than propose speculative hypotheses: Line 456-459: Whilst the molecular features that preclude ephrin-B3 utilization by CedV-G remain unclear, our structural hypothesis suggests that acquired ephrin-B1 specificity does not necessarily come at the expense of ephrin-B3 usage, as the LW motif is common to both ephrin-B2 and ephrin-B3.
Could the authors discuss how conserved EphrinB1 is among different species and whether CedV can transmit among species by utilizing EphrinB1?
Response: Ephrin-B1 is highly conserved amongst mammalian species (96-99% sequence similarity), at least equal to if not slightly less so than ephrin-B2. We have added in a statement with regards to ephrin-B1 conservation in the relevant part of the Discussion section: Line 472-475: Of note is the relatively high expression of ephrin-B1 in the lung, esophagus, and salivary glands ( Supplementary Fig. 5), which suggests that ephrin-B1 utilization could augment aspects of oropharyngeal transmission postulated for HNV [66], especially since ephrin-B1 is almost as conserved as ephrin-B2 across mammalian species (96-99% sequence similarity).
"As expected [43,54,55], ephrin-B2-Fc and ephrin-B3-Fc inhibited NiVpp entry into CHO-B2 cells, while ephrin-B1-Fc failed to strongly inhibit entry at concentrations as high as 10 nM (Fig. 5d,  If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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