Dear Dr. Liao,

Thank you very much for submitting your manuscript "Coronavirus endoribonuclease nsp15 induces host protein synthesis shutdown and ensures viral protein synthesis via evading PKR-eIF2α-mediated anti-viral translation shutoff" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Revisions should address the specific points made by each reviewer. We believe that these suggestions will help to improve your manuscript even further. 

In particular, please pay careful attention to reviewer concerns regarding the following:

• Cytotoxicity analysis for nsp15 overexpression
• Contradictory results when comparing the impact of nsp15 overexpression on exogenous and de novo protein synthesis inhibition
• The role for endonuclease activity in changes in host gene expression or localization of cellular signaling molecules  
• Contradictory results following infection and overexpression, including differences in localization of PABPC1 and the relevance of this protein in general
• The reproducibility and significance of protein synthesis inhibition as it relates to both the role of nsp15 endonuclease activity and the role of PKR. 
• Clarification of methods used to quantify puromycin labeling
• Conclusions that may overstate the data

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Emily Ledgerwood, PhD

Guest Editor

PLOS Pathogens

Ashley St. John

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

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: Non-structural proteins play an important role in the life cycle of viruses. These non-structural proteins play indispensable roles to suppress the physiological activity and immunity of host cells. In this paper, they studied showed the coronavirus endoribonuclease nsp15 inhibits host protein synthesis by targeting host RNA. This study revealed that nsp15 may regulate host protein expression by targeting host and viral RNA. While this result is somewhat convincing, a few concerns need to be addressed.

Reviewer #2: This resubmitted manuscript explores the role of CoV nsp15 in the induction of translational shutoff. While the role of nsp15 in reduction of viral dsRNA and prevention of PKR activation has previously been explored (Kinlder; Deng), the authors here show that this also holds true for another CoV, IBV. New findings, such as targets of nsp15 binding (including several RNA targets that regulated RNA expression) are novel, however some of the conclusions are overstated based on the data provided.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1.Fig1 A, please explain why there are the low expression level of GFP in the absence of an inhibitor. I have another concern whether the expression of β-actin protein will be inhibited, since nsp15 inhibits host protein synthesis. This problem was common in the description that follows. Fig1 B, the authors needed to indicate whether the protein is cytotoxic, since nsp15 inhibits host protein synthesis.

2.Fig4 showed some conflict with fig3. In the fig, the groups of MERS-CoV, SARS-CoV2 showed some ability of inhibition, however, the group of PDCoV showed weak ability of inhibition in protein synthesis. There are similar problem in S2 fig.

3.Fig8, Although it has been reported in the previous studies, how to make out nsp15 inhibit nuclear translocation of IRF3 in this fig.

Reviewer #2: - A number of times the authors state that differences in host gene expression and/or altered localization of signaling molecules (e.g. STAT) support the conclusion that nsp15 is targeting these host genes for degradation which is dependent on the endoU activity of nsp15. While this is a plausible hypothesis, this data alone is insufficient to support this. Alternative explanations could be that nsp15 is targeting other host RNAs that regulate expression of these proteins. This can be addressed by tempering these statements regarding what can be concluded from the data.

- The conclusion that nsp15 is important for host translational shutoff independent of PKR is appealing but unsubstantiated by the data (specifically Fig 11D). Relative quantitation of the signal in PKR siRNA kd cells infected with WT or mutant virus show 0.74 and 0.89 relative change in translation respectively. It's unclear if this difference is biologically (or statistically) significant, and it is unclear whether this quantitation data is from a single experiment or from multiple experiments. Moreover, it appears that the relative difference in puromycin incorporation in WT vs mutant infected cells (without siRNA treatment) is different in 11D vs 11B, where 11B shows substantial reduction following infection with both viruses, but 11D shows significantly more reduction in the mutant infected cells vs WT. While biological variation in such assays may be expected, it's challenging to know how consistent and biologically significant the phenotype in 11D is.

**********

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: 4.S1 fig, In the nsp15 expression group, why β-actin expressed in high level. And why some IBV protein not detected.

5.Fig2 A, why are NSP15 not detected in NSP15 transfection group. This description of the wild-type nsp15 is inaccurate, because there are only tranfection by NSP15 plasmid, but infection by virus.

6.Is the wild-type nsp15 transfeciton or viural infection? If it’s transfeciton, why the expression level was weak?

7.Fig5 B, The nuclear size varied greatly among the groups. Authors need to unify the scales.

8.Fig6, these viral nsp15 should indicated in the fig, rather than just the virus name, which can be misleading. And it's hard to make out the difference between these groups.

9.Fig11A, the labeling of these symbols(+,-) is confusing.

Reviewer #2: n/a

**********

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: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

PPATHOGENS-D-24-01388R1

Coronavirus endoribonuclease nsp15 suppresses host protein synthesis and evades PKR-eIF2 α -mediated translation shutoff to ensure viral protein synthesis

PLOS Pathogens

Dear Dr. Liao,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 pay particular attention to the following reviewer suggestions and give them due consideration. 

• Provide evidence to confirm that H1299 cells were successfully infected with wild-type viruses.
• Discuss the six viral transcripts and any relevant host proteins found to interact with nsp15 and relate those findings to the potential role of nsp15 in regulating viral gene expression during the viral life cycle.
• Clarify if nsp15 interacted with PABPC1 and relate to the proposed model. 
• If any of these interactions were validated, for instance by IP-WB, this should be stated.

Please submit your revised manuscript within 30 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Emily Ledgerwood, PhD

Guest Editor

PLOS Pathogens

Ashley St. John

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

Journal Requirements:

1) We notice that your supplementary Figures are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

2) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well. Currently, this grant number "32372999 " is missing from the Funding Information tab while this grant number "31772724" is missing from the Financial Disclosure field.

Please indicate by return email the full and correct funding information for your study and confirm the order in which funding contributions should appear. Please be sure to indicate whether the funders played any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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 addition to answering the reviewers' concerns, the author also supplemented RIP-Seq RNA data and GO and KEGG analyses. The paper contains a wealth of data.

Reviewer #2: This resubmitted manuscript explores the role of nsp15 in translational regulation. The authors have made significant experimental efforts to address some of the weaknesses in the originally submitted manuscript, including nsp15 interactome data.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: In the newly added RIP-Seq RNA data, the author mentioned infecting H1299 cells with wild-type viruses. Previous reports have shown that only the Beaudette strain of coronavirus infectious bronchitis virus after long-term domestication can infecte H1299 cell. Does the author have any evidence to confirm that the virus has successfully established an infection?

Reviewer #2: The inclusion of interactome data (RNA and protein is very interesting and provides additional support for some of the conclusions), however specific targets that were identified were not explicitly stated, only pathways and GO terms were used. This data would be even more convincing (and of interest to readers) if some discussion of specific targets were included and not simply relegated to the supplementary data file. For example, what were the 6 viral transcripts that were found to interact with nsp15? What does this potentially imply about the role of nsp15 in regulating expression of viral transcripts throughout the replication cycle? Regarding host protein interactions, was PABPC1 found to interact with nsp15? Were there other specific host proteins that were shown to interact with nsp15 that would further support the model proposed? Were some of these interactions validated using IP-WB?

**********

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: (No Response)

Reviewer #2: n/a

**********

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: Yes:  wenke ruan

Reviewer #2: 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.]

Figure resubmission:

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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Liao,

We are pleased to inform you that your manuscript 'Coronavirus endoribonuclease nsp15 suppresses host protein synthesis and evades PKR-eIF2 α -mediated translation shutoff to ensure viral protein synthesis' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

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

Best regards,

Emily Ledgerwood, PhD

Guest Editor

PLOS Pathogens

Ashley St. John

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

***********************************************************

Reviewer Comments (if any, and for reference):