PPATHOGENS-D-25-02465

Cetacean coronavirus spikes highlight S glycoprotein structural plasticity

PLOS Pathogens

Dear Dr. Hulswit,

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Félix A. Rey

Academic Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Ruben J.G. Hulswit, Tatiana Shamorkina, Joline van der Lee, Floor Rosman, Lisanne S Wetzels, Frank J.M. van Kuppeveld, Joost Snijder, Berend Jan Bosch, and Daniel L Hurdiss. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this manuscript, Hulswit et al. report high-resolution cryo-EM structures of two cetacean coronavirus spike proteins, showing that these spike trimers adopt a unique quaternary architecture, different from that of other coronaviruses. They have upright oriented S1B domains covered by S1A domains, and also a novel N-terminal S10 domain that resembles a cupin fold. Glycoproteomic analysis has identified a mucin-like loop at the trimer apex with extensive N-glycosylation and O-glycosylation contributing to the dense glycan shield that map help evade host immune responses. The authors suggest that these interesting structural features offer new insights into the evolutionary mechanisms for coronaviruses to expand their host range.

The manuscript is very well written and all the experiments are relatively straightforward and technically sound. These new structures indeed help advance our understanding of structure-function of diverse coronavirus spike proteins.

Reviewer #2: In this study, the authors determined high‑resolution cryo‑EM structures of spike glycoproteins from two cetacean gammacoronaviruses that infect marine mammals (beluga whale and bottlenose dolphin). Their structural analyses revealed an alternative quaternary assembly in which the S1B domains are tilted upright but shielded by extended loops from the S1A domain and a mucin-like apex, effectively concealing the conserved receptor-binding motifs. They further identified a novel ~200‑residue N‑terminal domain (S10) of host origin, structurally related to cupin proteins. Complementary glycoproteomic profiling showed that these spikes are heavily decorated with N‑glycans (>100 per trimer) and O‑glycans within the mucin‑like loop, forming a dense glycan shield. Together, these results demonstrate that cetacean coronaviruses have evolved unique structural and glycosylation features that expand the known diversity of coronavirus spikes, providing new insights into how these viruses adapt to novel hosts.

This is a well-written and straightforward manuscript that describes new insights into the diversity of coronavirus spike protein structure and architecture. The structural studies are performed to a high standard, and the analyses are thorough and thoughtful. Below are some minor comments and suggestions that may improve the manuscript.

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Part II – Major Issues: Key Experiments Required for Acceptance

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Reviewer #1: (No Response)

Reviewer #2: No major issues

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: There are only a few minor issues that should be addressed.

1. Discussion on the similarity between the S10 domain and cupin fold is not very convincing, as they do not look like having a same fold, at least, as shown in Fig.3d (perhaps a better view would help?). The best matches are of bacterial origin and show only 8-9% sequence identity (not sure whether there are any conserved motifs critical for the cupin fold). So trying to conclude that “the S10 sequence originates from either a mammalian or avian host given the observed species tropism of coronaviruses” sounds a bit stretchy.

2. In the figure legend –“Fig 1 SPA structure determination of BwCoV SED at 2.27 Å and BdCoV SED at 2.65 Å”, it seems to be the only place where SPA is mentioned and it should be spelt out (single particle analysis?) for general readers.

3. The sentences –“Neither hetero or local refinement appreciably improved the S10 density, presumably due to intrinsic local protein flexibility and abundance of (N- and O-) glycosylation sites on the surface of CeCoV S10 (described in detail later on) causing several S10 regions to be absent in the final atomic models (see Table S2 for a complete overview including unresolved S1A regions). The lack of observed conformational heterogeneity of domain S10 relative to the rest of the spike protein complex underlines the existence of a singular S10 state, packed against the S1A domain by a modest network of hydrogen bonds involving main and side chain atoms (Fig. 3c)” are a bit confusing for general readers. First, the S10 density could not be improved because of protein flexibility, and then, “lack of observed conformational heterogeneity of domain S10”. What they meant is probably that S10 domain is rigid relative to the trimer, but it has some disordered loops.

Reviewer #2: 1. The clashscores for the structures are a little high, particularly for the higher-resolution BwCoV S model (21.00). The authors may try to loosen the geometric constraints during refinement to improve the score.

2. The figures are, in general, clear and nicely illustrate the key points being made in the text. However, I feel Fig 1a could be improved. At a minimum, the EM maps could be removed so that readers can focus on the architecture of the spike. Improved coloring or surface representation may also help.

3. line 24. The zero in S10 needs to be superscripted.

4. Something seems off in lines 257-258: “Glycan densities were visible in the EM reconstructions for 26/37 N-unresolved regions of the maps.”

5. Out of curiosity, were the 2P stabilising mutations necessary? Beneficial?

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Hulswit,

We are pleased to inform you that your manuscript 'Cetacean coronavirus spikes highlight S glycoprotein structural plasticity' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Félix A. Rey

Academic Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):