Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor

Simone Queiroz Pantaleão; Lívia de Moraes Bomediano Camillo; Tainan Cerqueira Neves; Isabela de Godoy Menezes; Lucas Matheus Stangherlin; Helena Beatriz de Carvalho Ruthner Batista; Emma Poole; Michael Nevels; Eric Alisson Philot; Ana Ligia Scott; Maria Cristina Carlan da Silva

doi:10.1371/journal.pone.0277953

Peer Review History

Original SubmissionJune 16, 2022
18 Aug 2022 Decision Letter - Juliet V Spencer, Editor PONE-D-22-17017Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptorPLOS ONE Dear Dr. Carlan Silva, 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. Your manuscript has been reviewed by two experts whose comments are appended below. Overall the reviewers were enthusiastic about the manuscript and request only minor textual changes. Please address all the reviewers' comments in your rebuttal letter. Please submit your revised manuscript by Oct 02 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. 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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. 7. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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. 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: N/A 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: 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 study by Silva et al used computational methodology to perform molecular modeling of different vIL-10 isoforms with the IL-10R1. This interesting study highlights some intriguing aspects of vIL-10 biology that provides possible insight into the functions of different vIL-10 isoforms. - Text describing experiments shown in Figs 4&5 would be improved by more clearly describing the rationale behind focusing on isoform F. - Increased stability of Isoform B with IL-10R1 versus Isoform A is very curious. The discussion of this finding by the authors is good, however I think more explanation as to why Isoform A could still be more biologically active would improve the manuscript. What evidence or precedent is there that suggests that Isoform A intersection of binding sites 1 and 2 would enhance biological activity? - The manuscript could be improved overall by some editing, with paragraphs needing editing (e.g., removal/editing of very short paragraphs). Minor comments - - Line 315 Fig.2. Structure and aminoacid – needs a space - 321 – gray > grey - 552 - Therefore, despite of exhibiting – should read ‘in spite of’ Reviewer #2: In “Molecular Modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor” (PLoS ONE manuscript #: PONE-D-22-17017) Dr. Silva and colleagues identify a new spliced transcript variant of the CMV UL111a gene (cmvIL-10). This transcript was identified in MRC-5 cells and a human glioblastoma multiforme cell line infected with low-passage HCMV strain TB40E. The authors follow-up their finding using in silica experiments designed to predict protein tertiary structure and IL-10R1 binding, comparing the newly identified isoform H with the previously described cmvIL-10 isoforms. The strength of the report is in the very nice transcriptional and sequencing analysis followed up with nice molecular modelling. The impact of the study is somewhat limited due to the in silico approach used to assess potential cmvIL-10 isoform interaction with IL-10R1. Also, the authors suggest strain variation or PCR technique as possible explanations for the absence of cmvIL-10 isoform H in previous publications. It would be interesting to see the authors perform their transcriptional and sequencing analysis approach on cells infected with AD169 to directly address this issue, although I don't think it is a requirement for publication. Overall, the experiments are well designed and executed, and the proper controls are used throughout. The conclusions are supported by the experimental results. The manuscript is very well written, and all methods are described clearly and concisely. The main figures and layout are very nice as well, with only the very minor issue described below. This study will be of significant interest to scientists focused on the natural history of CMV, CMV host-pathogen interactions, and more broadly to immunologists interested in the IL-10 signaling cascade. Nice job! Minor issues: 1. The Y-axis label in Fig 5A is clipped ****** 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. https://doi.org/10.1371/journal.pone.0277953.r001
Revision 1
5 Oct 2022 Author Response -->General corrections -The manuscript was revised to match the PLOS ONE’s style requirements. Including the files names. -We included, in the financial disclosure, the following sentence. “The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”. -The Data availability statement has been modified. “The group chose not to use public repository. The data will be available upon request via direct contact with the corresponding author”. -We added the sequence identifier numbers of genbank in the manuscript. -The Orcid ID was associated with the corresponding author of the manuscript in the PLOS ONE system. -The gel and blot original images are available in the supplementary materials as a PDF file. -Legends of the supplementary materials are available in the final of the manuscript. -The bibliography has been revised -->Reviewer's Responses to Questions responses to the specific comments. -->Reviewer #1: - Text describing experiments shown in Figs 4&5 would be improved by more clearly describing the rationale behind focusing on isoform F. Response: We better described the focus in isoform F in the Results / Molecular docking section and Discussion. This isoform likely stablish the most stable complex having a consistent increasing in its RMSF number when in its complexed conformation. One can imagine that the ability to bind with higher affinity to the receptor could result in enhanced signalling or in competitive inhibition of binding by other isoforms to IL-10R1, therefore we focused on isoform F. - Increased stability of Isoform B with IL-10R1 versus Isoform A is very curious. The discussion of this finding by the authors is good, however I think more explanation as to why Isoform A could still be more biologically active would improve the manuscript. What evidence or precedent is there that suggests that Isoform A intersection of binding sites 1 and 2 would enhance biological activity? Response: As far as we know there is no precedent evidence suggesting that intersection of binding sites 1 and 2 enhances the biological activity of isoform A. According to our data we suggest that this may occur. We can observe that isoform sites 1 and 2 in isoform A are very close to each other, possibly forming a large site, favouring connections and improving the binding possibilities. We describe it better throughout the text, mainly in the results section. - The manuscript could be improved overall by some editing, with paragraphs needing editing (e.g., removal/editing of very short paragraphs). Response: We performed a removal of short paragraphs and textual corrections. -Minor comments - Line 315 Fig.2. Structure and aminoacid – needs a space - Corrected - 321 – gray > grey - Corrected - 552 - Therefore, despite of exhibiting – should read ‘in spite of’ - Corrected -->Reviewer #2: The impact of the study is somewhat limited due to the in-silico approach used to assess potential cmvIL-10 isoform interaction with IL-10R1. Also, the authors suggest strain variation or PCR technique as possible explanations for the absence of cmvIL-10 isoform H in previous publications. It would be interesting to see the authors perform their transcriptional and sequencing analysis approach on cells infected with AD169 to directly address this issue, although I don't think it is a requirement for publication. -Minor issues: 1. The Y-axis label in Fig 5A is clipped - Corrected Attachments Attachment Submitted filename: Rebutal Letter.pdf https://doi.org/10.1371/journal.pone.0277953.r002
10 Oct 2022 Decision Letter - Juliet V Spencer, Editor PONE-D-22-17017R1Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptorPLOS ONE Dear Dr. Carlan Silva, 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. Thank you for your attention to most of the reviewer comments. However, there are still multiple "paragraphs" in your discussion that consist of 1 or 2 sentences. The reviewers asked you to correct this and it has not been done. A single sentence is not a paragraph. Please seek assistance with editing from a native English speaker and pay careful attention to paragraph structure. Please submit your revised manuscript by Nov 24 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, Juliet V Spencer, Ph.D. 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: [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. https://doi.org/10.1371/journal.pone.0277953.r003
Revision 2
2 Nov 2022 Author Response The response to specific reviewer and editor comments are available in the attach files too. Reviewer #1: - Text describing experiments shown in Figs 4&5 would be improved by more clearly describing the rationale behind focusing on isoform F. Response: We better described the focus in isoform F in the Results / Molecular docking section and Discussion. This isoform likely stablish the most stable complex having a consistent increasing in its RMSF number when in its complexed conformation. One can imagine that the ability to bind with higher affinity to the receptor could result in enhanced signalling or in competitive inhibition of binding by other isoforms to IL-10R1, therefore we focused on isoform F. - Increased stability of Isoform B with IL-10R1 versus Isoform A is very curious. The discussion of this finding by the authors is good, however I think more explanation as to why Isoform A could still be more biologically active would improve the manuscript. What evidence or precedent is there that suggests that Isoform A intersection of binding sites 1 and 2 would enhance biological activity? Response: As far as we know there is no precendent evidence suggesting that intersection of binding sites 1 and 2 enhances the biological activity of isoform A. According to our data we suggest that this may occur. We can observe that isoform sites 1 and 2 in isoform A are very close to each other, possibly forming a large site, favouring connections and improving the binding possibilities. We describe it better throughout the text, mainly in the results section. - The manuscript could be improved overall by some editing, with paragraphs needing editing (e.g., removal/editing of very short paragraphs). Response: We performed a removal of short paragraphs and textual corrections. Minor comments - Line 315 Fig.2. Structure and aminoacid – needs a space Corrected - 321 – gray > grey Corrected - 552 - Therefore, despite of exhibiting – should read ‘in spite of’ Corrected Reviewer #2: The impact of the study is somewhat limited due to the in-silico approach used to assess potential cmvIL-10 isoform interaction with IL-10R1. Also, the authors suggest strain variation or PCR technique as possible explanations for the absence of cmvIL-10 isoform H in previous publications. It would be interesting to see the authors perform their transcriptional and sequencing analysis approach on cells infected with AD169 to directly address this issue, although I don't think it is a requirement for publication. Minor issues: 1. The Y-axis label in Fig 5A is clipped Corrected General corrections: -The manuscript was revised to match the PLOS ONE’s style requirements. Including the files names. -We included, in the financial disclosure, the following sentence. “The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”. -The Data availability statement has been modified. The data is available in https://www.kaggle.com/datasets/tainanneves/data-availability-hcmv-il10-molecular-models -We added the sequence identifier numbers of genbank in the manuscript. -The Orcid ID was associated with the corresponding author of the manuscript in the PLOS ONE system. -The gel and blot original images are available in the supplementary materials as a PDF file. -Legends of the supplementary materials are available in the final of the manuscript. -The bibliography has been revised Attachments Attachment Submitted filename: Rebutal Letter.docx https://doi.org/10.1371/journal.pone.0277953.r004
8 Nov 2022 Decision Letter - Juliet V Spencer, Editor Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor PONE-D-22-17017R2 Dear Dr. Carlan Silva, 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, Juliet V Spencer, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0277953.r005
Formally Accepted
15 Nov 2022 Acceptance Letter - Juliet V Spencer, Editor PONE-D-22-17017R2 Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor Dear Dr. Carlan da Silva: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Juliet V Spencer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277953.r006

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