PONE-D-22-23172Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cellsPLOS ONE

Dear Dr. Cho,

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 submit your revised manuscript by Nov 26 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,

Saeid Ghavami, PhD

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 noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://pubmed.ncbi.nlm.nih.gov/34668777/?

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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

"No authors have 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.

4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Additional Editor Comments:

Dear Professor Kyoung-Oh,

Thank you for submission to PLOS ONE and I sincerely do apologize for late processing of your article. It was very hard to secure good reviewer and get feedback and comments. Besides addressing the respected reviewers,

1- Please use Necrostatin and address its effect in your model.

2- Please provide original WB without any crop.

Thank you

Saeid Ghavami, PhD, AE

[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: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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: 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: The present manuscript ‘Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells’ describes an interesting work and has potential to become valuable reading materials. However, there are several minor concerns and confused questions before it can be published by PLOS ONE.

Minor concerns:

1. The P values for all graph statistics are incorrect and the number of ‘*’ on the picture is also very weird, newly statistical analysis is recommended.

2. The full and abbreviated names of proper nouns are confusing. It is recommended that the full name only appear once.

3. Immunoprecipitation assay: line 237-239, check the writing of ‘°C’.

4. Page 37, line 653, replace “TCID50” with “TCID50”

5. Please check for grammar errors, for example line 720 and line 721.

6. Figure 6E and 6F, please explain why the similar bands in the Z-VAD-FMK treatment group in Fig. 6E have a statistically significant darkening trend in Fig. 6F.

7. Figure 8D, PSaV+ Z-VAD-FMK treatment group, please explain why the higher the concentration of Z-VAD-FMK, the better the cell viability, why both anti- necroptosis and pro-necroptosis can improve cell activity, isn't this contrary to your conclusion in this article?

8. Abstract: “Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating antiviral action of PSaV-induced apoptosis.” Your data may demonstrate a proviral effect of necroptosis, but no direct data suggest an antiviral effect of apoptosis, please explain more directly how you came to this conclusion.

Reviewer #2: Title: Porcine sapovirus-induced RIPK1- dependent necroptosis is proviral in LLC-PK cells

Author: Sharif et al

The manuscript shows that Porcine Sapovirus (PSaV) induces necroptosis and apoptosis in the Porcine Kidney Cells (LLC-PK). Inhibition of critical regulatory and executioner proteins of necroptosis with their respective chemical inhibitors (Nec-1, GSK'872, NSA) or silencing those three proteins (RIPK1, RIPK3, and MLKL) with siRNA prevented the phosphorylation of the proteins and virus replication and targeted viral proteins. Inhibition of necroptosis during virus infection increases cell viability relative to the virus-treated cell alone, and inhibition of apoptosis increases necroptosis and promotes virus replication. The authors propose a counterbalance between apoptosis and necroptosis in virus-infected cells.

Major

1) Fig 1, 2, 3, 8, and 9. The data shows that virus infection kills the cells irrespective of whether both necroptosis and apoptosis are inhibited, and remanding cells seem to exhibit both necroptosis and apoptosis characteristics. Please comment on this phenomenon.

2) Figure 6F and G. On page 15, line 314, it reads, "Interestingly, inhibition of necroptosis by the treatment with each chemical necroptosis inhibitor suppressed the expression of PSaV VPg protein (Fig6E and 6F) and PSaV titer (Fig 6G)." Please explain why there is a difference in the protein level produced by the virus (VPg in Figure 6F) but not necessarily in the amount of virus replication measured by the TCID50 (Fig6G).

3) line 316: "However, treatment with chemical apoptosis inhibitor reduced viral protein expression and titer (Fig 6E-G)." The caspase inhibitor increased the viral protein at 24hrs and the PSaV titer at 36 hours relative to the PSaV treatment only in those time points. Please clarify and discuss the significance.

4) Fig 6F and Fig 6G as in other figures? Please show the mock control for these experiments. What is the expected cell viability for the LLC-PK cell not treated with the PSaV?

5) Necrosis vs. Necroptosis.

On page 16, line 326, the authors wrote, "A shift in cell death modality from apoptosis to necrosis and vice versa has been observed in response to human immunodeficiency virus type-1 (HIV-1), Theiler's murine encephalomyelitis virus (TMEV), and rotavirus infections in the presence of chemicals against the necroptosis or apoptosis." Necrosis is an unregulated, uncontrolled, and irreversible cell death process. There is swelling of the organelles, plasma membrane rupture, and eventual lysis of the cell. Necrosis is triggered in the context of excessive external stress, such as heat, ischemia, and pathogen infection. It doesn't need the Tumor Necrosis Factor Receptor activation or a specific cell death complex to trigger it. Zhou W, Yuan J. Semin Necroptosis in health and diseases. Cell Dev Biol. 2014 Nov;35:14-23. Festjens N, Vanden Berghe T, Vandenabeele P. Necrosis, a well-orchestrated form of cell demise: signaling cascades, important mediators and concomitant immune response. Biochim Biophys Acta. 2006 Sep-Oct;1757(9-10):1371-87. In contrast, "necroptosis is a more physiological and programmed type of necroptotic death and shares several key processes with apoptosis. It occurs due to activating the kinase domain of the receptor-interacting protein 1 (RIP1) and the assembly of the RIP1/RIP3-containing signaling complex. It is triggered by tumor necrosis factor (TNF) family members, needs caspase eight inhibition, and assembly of necrosome (RIPK1-RIPK3 complex IIb) with activation of MLKL." Elmore SA, Dixon D, Hailey JR, Harada T, Herbert RA, Maronpot RR, Nolte T, Rehg JE, Rittinghausen S, Rosol TJ, Satoh H, Vidal JD, Willard-Mack CL, Creasy DM. Recommendations from the INHAND Apoptosis/Necrosis Working Group. Toxicol Pathol. 2016 Feb;44(2):173-88. Even though necroptosis is classified as a type of necrosis, it would be better to use the term necroptosis in that sentence because necrosis cannot be shifted to apoptosis, and apoptosis cannot be shifted to necrosis. Even the four citations at the end of the sentence (47, 53, 55, 56) have necroptosis in their tittles to convey the message that necroptosis is the process that can be shifted to apoptosis.

6-) In figure 8, it could be appreciated that the cells treated with PSaV die anyways, irrespective of the inhibitor used to prevent apoptosis of necroptosis. Therefore, contrary to the line on page 16, line 334, which reads, "Interestingly, necroptosis inhibition of PSaV-infected cells by the individual treatment with the inhibitor against the RIPK1, RIPK3, or MLKL showed an increase in cell viability (Fig 8A-C) and the proportion of apoptotic cells." I suggest describing the findings in Figure 8, stating that inhibiting apoptosis or necroptosis did not prevent cell death by PSaV but lessened its impact. It's evident that by 36hrs, all of the treatments in Fig 8 show cell viability at less than 50%.

7-) In Figure 9, the same observation can be made for Fig 8. Even when it is true that inhibiting necroptosis and apoptosis changes one cell death process into another, the overall trend is that both processes are increased and that, irrespective of the treatment, the cells will show increased necroptosis or necroptosis. A better way of describing the findings in Figure 9 is to say that inhibiting necroptosis or apoptosis changes the proportion of cells passing through apoptosis or necroptosis.

Minor

1) In Figures 1C, 2C, 6F, 6G, 8ABCD, and 9ABCD. Did the author try using two-way ANOVA? These figures show more than two independent categorical variables.

**********

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: 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.

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

PONE-D-22-23172R1

Dear Dr. Cho,

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,

Saeid Ghavami, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

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 #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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 #1: Yes

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 #1: Yes

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 #1: (No Response)

Reviewer #3: The revision significantly improves the paper. The authors have carefully addressed all the comments in a proper way.

**********

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 #1: No

Reviewer #3: Yes: Saeid Ghavami

**********