PONE-D-21-14234

1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution.

PLOS ONE

Dear Dr. Agback,

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 be aware that both reviewers raised a significant number of concerns that should be completely addressed in a revised version of the manuscript, which will likely undergo a second round of review. Of particular importance is to clarify the novelty of the work, in relation to previous publications.

Please submit your revised manuscript by Jul 25 2021 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: http://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,

Oscar Millet

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 you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

3. We note that Figure 1 and Figure 2 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 and Figure 2 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

Additional Editor Comments (if provided):

[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

**********

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

Reviewer #1: Yes

Reviewer #2: 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

**********

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: No

**********

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: The manuscript describes the NMR chemical shfit assignment of the nsp1 protein from SARS-CoV-2. Unfortuneatly, it is the second report of this NMR resonance assignment. The authors rightly reference the previous work, but the differences e.g. construct design is not being discussed. The authors do not compare their chemical shift assignment with the previously published data.

The authors here also investigate mutants of nsp1, but the differences e.g in the dynamics of "wildtype" and mutant protein are not presented.

At this point, I cannot recommend publication of this work.

Reviewer #2: The submitted manuscript presents a far-reaching NMR analysis of the SARS-CoV-2 Nsp-1 protein, using both wild-type and two mutant proteins, and at two temperatures. For this, the authors employ a very complete and high-level set of NMR experiments, going beyond the standard protocol and addressing (by MUSIC type experiments) the specific needs for proteins with large unstructured regions (as in Nsp-1). Thus, the work deserves publication for its sound technical implementation and for the high relevance of the targeted protein. However, the manuscript requires a minor-to-mayor overhaul for the reasons listed below, where especially one critical prior work (correctly cited as ref. 31) must be considered more thoroughly. Of note, while this prior work formally limits the novelty of the presented manuscript, this does not constitute a valid reason for rejection in my eyes - rather, I "merely" request a more detailed comparison (stressing the agreements and working out the minor differences) to consolidate, deepen, and extend our understanding of this functionally important protein.

General requests:

- I recommend that a native English speaker revises the manuscript as it contains awkward wording in several larger parts of the text (e.g., legend of Fig. 1, text paragraphs l. 211-217, l. 269-272, 320-324, 334-336) that strongly compromises readability.

- The cited reference [31] reports critical prior work and must be considered in more detail. Thus, the authors should work out more clearly, and wherever relevant in the manuscript, the main differences and agreements between their new work and ref. [31]. For instance: For which further residues were the authors able to provide a NMR characterisation? Why do the authors find slightly different residue segments for the unstructured N- and C-terminal and for the structure central regions? Why could the authors not confirm the short C-terminal helix H3? Could such differences be due to their use of an older version of TALOS (TALOS+ instead of TALOS-N), or to the slightly different experimental conditions (pH, temperature, etc.)?

Specific requests:

- Table 1 (NMR parameters): Since recorded FID resolution is the inverse of the maximal sampling time (i.e., FID resolution = 1/AQ, which should be stated clearly in the caption to column 2), very low F1 and F2 resolutions follow from the listed data and I wonder whether there might be a systematic error (factor 2?) in these numbers? For instance, the listed 12.3 ms for 15N result in only 81 Hz = 1.35 ppm resolution. Such low resolution makes little sense especially for intrinsically disordered proteins/regions (as present in Nsp-1). Moreover, it invalidates both resolution enhancing NMR methods adopted by the autors, i.e. non-uniform sampling (NUS) and semi-constant t1(15N) evolution. Already constant-time evolution of 1J(NCO) = 15 Hz or 1J(NCA) = 9-12 Hz demands transfer delays at least twice as long as the cited 12.3 ms (i.e., up to 30 ms), and semi-constant time evolution would require even longer delays. Please clarify this obvious contradiction. I also suggest to add a further column to Table 1 listing the pertaining total measurement time for each experiment.

- L. 134: state that 14.1 T correspond to 600 MHz for 1H to enable conversion of resolution, from Hz to ppm (as required to convert the maximal evolution times into recorded FID resolutions, see my notes on Table 1 above).

- L. 175/176: It is stated that folded and disordered regions show distinct NMR relaxation properties exacting distinct optimal “NMR experimental conditions”. This should be better elaborated. For instance: lower temperatures favour NMR on IDP due to reduced HN/H2O exchange while higher T favour folded regions due to reduced T2 relaxation. Mention further NMR parameters like transfer and interscan delays that also differ – how was this implemented or considered?

- Terminology: “DIPSI2-N-HSQC” and “MLEV17-C-HSQC” (line 141) are unconventional names (taken directly from the BRUKER pulse program library?) – better use the standard NMR experiment name = TOCSY-HSQC (or was it a HSQC-TOCSY?). Also, in line 143, I suppose the authors meant to say “NOESY-HSQC” (or HSQC-NOESY)?

- L.143: HA assignment should, in principle, follow directly from a combination of HACACO with HNCO and HN(CO)CA spectra, making the acquisition of both NOESY-HSQC meaningless and superfluous for this purpose. I suppose, however, that these latter spectra were rather recorded with the objective to search for structure indication? If so, please indicate!

- The optional addition of MgCl2 (2.5 mM) is unclear to me and should be explained. How did it affect the NMR spectra?

- The abbreviation “aa” should be avoided and spelled out: “amino acid”, or “residue”

- The short helix a3 appears to be missing in the NMR structure, but shows indication of restricted mobility (lines 309 – 312). As a matter of fact, the X-ray structure rather reports a short 3-10 (instead of canonical alpha) helix here. Thus, have the authors considered the possible presence of a 3-10 helix? What secondary structure does the CSI for the HA nuclei suggest (considering that helicity indicated only by HA may suggest a 3-10 helix)?

- Terminology: While technically correct, the term “cross peak” for an HSQC signal appears very confusing as it is conventionally reserved to NOE cross peaks. This may give rise to confusions, for instance, when reading through lines 328-330 where the disordered regions of Nsp-1 are discussed, but where no (NOE) cross signals are expected.

- This confusion is accentuated by the subsequent conclusion of the authors (lines 330-332, and section “Conclusion”) that folded and unfolded regions in Nsp-1 would show some kind of intramolecular interactions. As it stands, this conclusion is not substantiated enough (although the prior work – ref. 31 – also suggested this from the notable solubility enhancement of the C-terminus on the folded domain).

- I was intrigued by the finding of a double set of signals for residues 122-125 (line 221). Then, however, I wonder which of both signal shifts (for each residue) was used for the secondary structure analysis shown in Fig. 3b, and how the predicted secondary structuredness differs for both sets of shifts? This should be clarified and the alternative signal shifts should be reported more clearly – ideally, by inclusion into Fig. 3b (e.g., as a second set of coloured bars). Apparently, there is someconformational dynamics towards the C-terminus of strand b7: could this possibly be related to a “kink” in this strand, near V121? The competing work in ref.[ 31] also mentions such conformational heterogeneity (without further specifying the residues) and suggests a possible role of cis/trans isomerism at a proline residue (again, without specifying the residue number, but possibly referring to P115). Interestingly also, the RCI-S2 order parameters derived by the authors and in ref. [31] differ more strongly in exactly this region (res. 120 – 130), where the authors derive a significantly higher rigidity than reported in [31]. I would greatly welcome if the authors provided more details and some deeper discussion of this interesting observation of conformational exchange exactly in the transition region between folded and unfolded (C-terminal) regions, as this might even have a relevance for the known “switch” in Nsp-1 function (from host translation inhibition to viral translation initiation).

**********

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: Tammo Diercks

[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.

1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution.

PONE-D-21-14234R1

Dear Dr. Agback,

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,

Oscar Millet

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: