PONE-D-22-24483Inside-out: antibody-binding reveals potential folding hinge-points within the SARS-CoV-2 replication co-factor Nsp9PLOS ONE

Dear Dr. Littler,

Thank you for submitting your manuscript to PLOS ONE. I also apologize for my tardiness in getting your manuscript reviewed. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

       My recommendation for major revision is based on the comments from one of the reviewers. However, I believe these revisions are feasible.

        Please submit your revised manuscript by Dec 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Massiah

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating in your financial disclosure:  

"The work is funded by internal Departmental support." 

PLOS ONE requires you to include in your manuscript further information about the funder so that any relevant competing interests can be assessed. Please respond to the following questions:

a) Please state whether any of the research costs or authors' salaries were funded, in whole or in part, by a tobacco company (our policy on tobacco funding is at http://journals.plos.org/plosone/s/disclosure-of-funding-sources)  

b) Please state whether the donor has any competing interests in relation to this work (see http://journals.plos.org/plosone/s/competing-interests) . 

c) Please state whether the identity of the donor might be considered relevant to editors or reviewers’ assessment of the validity of the work.

d) If the donors have no perceived or actual competing interests, please state: “The authors are not aware of any competing interests”. 

This information should be included in your cover letter. We will amend your financial disclosure and competing interests on your behalf.

4. Thank you for stating the following in your Competing Interests section:  

"There are no competing interests."

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

5.In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

6. Please amend your authorship list in your manuscript file to include author "Yue Pan".

7. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 

8. Please upload a copy of Supporting Information Table 1 which you refer to in your text on page 3.

9. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript reports a structure of SARS-CoV-2 nsp9 bound to nanobody VHH2NSP23. Identification of the binding epitope residues will aid future drug development. Changes in the GxxxG helix, which is responsible for Nsp9-NiRAN interactions, moving to the cavity between the s2-s3 and s4-s5 loops, are particularly noteworthy. Understanding the Nsp9/NiRAN interactions is essential to solving the CoV-2 capping mechanism. Although several models have been proposed, the alternative structure of nsp9 revealed in this study may open new possibilities for research. For example, are there native binding partners of nsp9 that can induce these conformational changes? If yes, will it change the capping pathway?

The manuscript is clearly written and well illustrated. I suggest a few minor changes

1) Page 2 line 4, change ‘Nsp12’ to ‘nsp12’. All viral nsps should be in lowercase throughout the manuscript.

2) Page 3 line 9, active NMPylation can be supported by Mg2+. Ref “NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain, Nucleic Acids Research, https://doi.org/10.1093/nar/gkab677”. Line 26, I suggest deleting ‘compromising binding’ here since no data is supporting it in this manuscript. Lines 41-43 are very confusing, please rewrite the sentence. Maybe separate it into two sentences.

3) Page 4 lines 13 – 14, does this description correspond to Figure 1C?

4) Page 7 line 3, if the issue of ‘We note that crystallization conditions can be unusual’ was solved by HSQC, this sentence can be deleted. line 14, 6 hours. line 15, 50 mM, please check other places. Line 16, in the presence of buffer A? line 35, ‘1:0.5 ratio’? It’s better to specify the ratio of what to what.

5) ‘VHH2NSP23’ or ‘VHH’? Please choose one and stick to it.

6) Are ‘nsp9’ and ‘nsp9COV19’ different? The use of them is somewhat confusing.

7) Ref 21 has been published.

8) The names in ‘Author Contributions’ part don’t match the authors in the titles.

9) Figure 1 legend, buried surface area (BSA); what’s ‘germ’? where is the BSA of CDR1? And please change the color of CDR1 since green is occupied by nsp9. Use ‘kDa’ for the marker of the SDS-PAGE in (A). Not mandatory, but I suggest redoing the SDS-PAGE if possible. The nsp9 band can barely be seen. In (C), change CDR2 residues to blue.

10) In figure 2(A), not all of these colored key residues are labeled. (B) the ‘s6’ has a different font. (D) what’s the meaning of the color gradient? (E) what’s the meaning of the underlines?

Reviewer #2: Pone-D-22-24483

A paper describing the structure of SARS-CoV-2 nsp9 complexed with a nanobody. Nsp9 undergoes a rearrangement with the nanobody binding, disrupting a conserved Beta-sheet and creating new alpha-helices in an inside out pattern. The area changed by the nanobody is thought critical to nsp9 function in the initial viral-mRNA capping reaction, and the authors speculate that this interruption of nsp9 function could be therapeutic in nature. They also speculate that the novel structure could be a naturally occurring as a structural change that occurs in nsp9 during its functions during viral infection. A few comments to address on revision

1. The authors speculate that antibodies or nanobodies that have this function of binding nsp9 and inducing the change in structure could be therapeutic during viral infection. They mention in example, therapeutic antibodies that are against spike. I think these anti-nsp9 nanobodies or antibodies would have to delivered intracellularly and make it to the double membrane enclosed replication complex of SARS-CoV-2. I think the authors should point this out and give ideas for such intracellular delivery.

2. Line 32 on page 5, “guild” should probably be “guide”

Reviewer #3: Comments to authors:

In this manuscript, Pan et al. characterized the interactions between SARS-CoV-2 Nsp9 protein with an existing anti-Nsp9 nanobody using X-ray crystallography. The authors solved the crystal structure of the Nsp9-nanobody complex at 2.4 Å resolution. They confirmed that the binding interactions are centered around a Trp-53 within the Nsp9, consistent with the previous NMR-based structural studies of the same complex. They also found that binding the nanobody brings conformational changes in the Nsp9, hampering the Nsp9 binding with its natural substrate NiRAN domain of Nsp12. However, the manuscript has not explicitly discussed the mechanism of such conformational change, which may be a consequence of the nanobody binding.

The manuscript is well-written and presents an important advancement in the structural understanding of SARS-CoV-2 proteins and their complexes with antibodies, which may help develop alternative strategies for COVID-19 prevention and control. Broadly, conclusions are supported by the experiments, but several points are discussed poorly. For example, the authors said that “the new VHH-bound architecture of Nsp9COV19 represents a novel fold”, but it is unclear how and why it is a “novel” fold of a protein. I expect the authors consider the following points to revise the manuscript before it can be accepted for publication.

1) VHH should be defined when used first-time in the text. It is unclear until reading down to the results section.

2) Fig.1A: The labels on top of the bands make them difficult to see. I assumed these were the MW markers.

3) The crystallographic asymmetric unit contained two complexes. It is unclear whether these complexes tend to dimerize in solution. Some tests, such as SEC or native gel electrophoresis, will be helpful.

4) What is the affinity of Nsp9 binding with VHH? How does it differ from Nsp9 binding with the NiRAN domain? It may give a clearer perspective on the energetics of Nsp9 deformations caused by the VHH binding.

5) Two Nsp9 molecules contact each other heavily within the crystallographic asymmetric unit. Does Nsp9 dimerize in the solution? Does this contribute to the observed deformation of the Nsp9 upon VHH binding? It would be helpful to include this discussion.

6) The authors discussed that Nsp9 Trp-53 is the primary binding site for the VHH. However, they did not discuss how Trp53 mutation impacts this binding.

7) A figure with crystallographic B-factors will be helpful to see rigid and more dynamic regions in the Nsp9-VHH complex.

8) Crystallographic data collection and refinement statistics table should be presented (in the manuscript or the SI).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Wesley C Van Voorhis

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-24483R1Inside-out: antibody-binding reveals potential folding hinge-points within the SARS-CoV-2 replication co-factor Nsp9PLOS ONE

Dear Dr. Littler,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

  Please note that despite the reviewer agreeing that the revise manuscript is acceptable, there are concerns that the reviewer feels that the authors did not adequately address or address suitable.

Please submit your revised manuscript by Apr 06 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Massiah

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: Authors have addressed most of the issues raised in previous version. However, some of the things authors said "done" in the response letter are not reflected in the revision. For example, the Fig. 1A still contains labels on top of the bands.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Inside-out: antibody-binding reveals potential folding hinge-points within the SARS-CoV-2 replication co-factor Nsp9

PONE-D-22-24483R2

Dear Dr. Littler,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michael Massiah

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: