Future Sequon Finder - A novel approach for predicting future N-linked glycosylation sequon locations on viral surface proteins

Shane P. Bryan; Martin S. Zand

doi:10.1371/journal.pone.0328174

Peer Review History

Original SubmissionApril 14, 2025
12 May 2025 Decision Letter - Cheorl-Ho Kim, Editor PONE-D-25-18727Future Sequon Finder - A novel approach for predicting future N-linked glycosylation sequon locations on viral surface proteinsPLOS ONE Dear Dr. Zand, 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 Jun 26 2025 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. 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Thank you for stating the following financial disclosure: “The project described in this publication was supported by the University of Rochester CTSA award number UL1 TR002001 from the National Center for Advancing Translational Sciences (MZ), and award number R01 AI 134058 from the National Institute for Immunology, Allergy, and Infectious Diseases from the National Institutes of Health (MZ, SB) . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. 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. 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. Additional Editor Comments : Dear Dr Zand, Thank you for your submission of your interesting study to Plos One. I have completed the review process and am pleased to inform that your manuscript can be accepted for publication after your minor revision. As you read, you can easily and simply revise the original manuscript. Thank you for your submission. Looking forward to receiving your revision. Cheorl-Ho Kim, Ph.D Editor [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: Yes ****** 3. 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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: (1) The authors mainly consider the exposure of newly acquired N-glycosylation sites on the molecular surface as an indicator of functional relevance. However, it is also important to note that the formation of the HA trimer is essential for glycan-binding activity. Since trimerization is distinct from immune surveillance mechanisms, it may impose constraints on further molecular evolution. This aspect should be discussed in the Discussion section of the manuscript. (2) It is generally considered that N-linked glycosylation enables viruses to evade immune surveillance mechanisms. Has the efficacy of this mechanism been evaluated using other viruses? And if there are issues to be considered in the program, what aspects should be taken into account? On this point, the authors shold include their opinions in the main text, which would provide valuable information for readers conducting similar studies. Reviewer #2: This manuscript by Zand et al. titled describes a program (Future Sequon Finder) to predict where N-linked glycosyltion sites will likely emerge on viral surface proteins, tested primarily on influenza hemagglutinin. This paper addresses a relevant and timely problem, but several concerns need to be addressed before it can be accepted for publication. - The program uses genetic data; however, the method by which the DNA sequence is translated into a protein sequence remains unclear. There are six open reading frames, and it is uncertain how the program will determine the correct open reading frame. - The proposed program predicts the likely location for glycosylation; however, it does not guarantee that this site will indeed undergo glycosylation. While the exposure of the side chain increases chances of potential glycan attachment, other factors also influence whether glycosylation will occur or not. Can authors comment on that? - Author did not consider oligomerization of protein in when calculation exposed potential glycosylation sites. Could it be a limitation of the program. - The definition of a sequon should be clearly stated. Given that not all glycosylation sites conform to the Asn-X-Ser/Thr rule, the specific amino acid sequences the program looks for are required. - Authors should provide a access to the program if possible. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
25 Jun 2025 Author Response We have made the requested corrections, specifically: 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 We have changed the file names of our supplemental figures and we have updated the formatting of the author affiliations to be in accordance with PLOS One’s formatting guidelines. 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. In accordance with PLOS ONE's guidelines, we have created a publicly accessible GitHub repository containing all author-generated code used in this study. To ensure long-term accessibility, we have archived the repository with Zenodo and generated a DOI. The DOI has been cited in the manuscript. 3. Thank you for stating the following financial disclosure: “The project described in this publication was supported by the University of Rochester CTSA award number UL1 TR002001 from the National Center for Advancing Translational Sciences (MZ), and award number R01 AI 134058 from the National Institute for Immunology, Allergy, and Infectious Diseases from the National Institutes of Health (MZ, SB) . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. This statement has been added. 4. 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. The captions for our Supporting Information files have been adjusted to match PLOS One’s formatting requirements. Optional figure legends have been omitted due to the self-explanatory nature of the file names. 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. We have removed several references that are no longer cited directly in the text, or are better represented by another reference already in the bibliography. The titles of the removed references are as follows: 1. Cited manuscript: Origin of the pandemic 1957 H2 influenza A virus and the persistence of its possible progenitors in the avian reservoir (Schafer) Reason removed: The section containing the reference was removed. 2. Cited manuscript: The re-emergence of H1N1 influenza virus in 1977: a cautionary tale for estimating divergence times using biologically unrealistic sampling dates (Wertheim) Reason removed: The section containing the reference was removed. 3. Cited manuscript: The Reemergent 1977 H1N1 Strain and the Gain-of-Function Debate (Rozo) Reason removed: The section containing the reference was removed. 4. Cited manuscript: Influenza Research Database: An integrated bioinformatics resource for influenza virus research (Zhang) Reason removed: This is a reference to the Influenza Research Database, which had joined with the BV-BRC prior to writing this manuscript. Given that the data used in this study was obtained from the BV-BRC, all references to the IRD were replaced with references to the BV-BRC. Additionally, several references were added to support new text in the revised manuscript. The titles of the added references are as follows: 1. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding (Petrescu) 2. N-glycosylation efficiency is determined by the distance to the C-terminus and the amino acid preceding an Asn-Ser-Thr sequon (Baño-Polo) Review Comments to the Author Reviewer #1: (1) The authors mainly consider the exposure of newly acquired N-glycosylation sites on the molecular surface as an indicator of functional relevance. However, it is also important to note that the formation of the HA trimer is essential for glycan-binding activity. Since trimerization is distinct from immune surveillance mechanisms, it may impose constraints on further molecular evolution. This aspect should be discussed in the Discussion section of the manuscript. Several sentences have been added in the manuscript to address the trimerization of the HA. (2) It is generally considered that N-linked glycosylation enables viruses to evade immune surveillance mechanisms. Has the efficacy of this mechanism been evaluated using other viruses? And if there are issues to be considered in the program, what aspects should be taken into account? On this point, the authors should include their opinions in the main text, which would provide valuable information for readers conducting similar studies. The final paragraph of the manuscript has been modified to address the first point by elaborating on the extent to which spike proteins are glycosylated in SARS-CoV-2, and the third-to-last paragraph of the manuscript has been modified to address the second point by elaborating on the weaknesses of the current approach. Reviewer #2: This manuscript by Zand et al. titled describes a program (Future Sequon Finder) to predict where N-linked glycosyltion sites will likely emerge on viral surface proteins, tested primarily on influenza hemagglutinin. This paper addresses a relevant and timely problem, but several concerns need to be addressed before it can be accepted for publication. - The program uses genetic data; however, the method by which the DNA sequence is translated into a protein sequence remains unclear. There are six open reading frames, and it is uncertain how the program will determine the correct open reading frame. The following sentences have been added to the manuscript: “Sequences were curated to ensure they began with the correct start codon, eliminating reading frame ambiguity for translation. Sequences that did not begin with a start codon were eliminated.” This curation was performed by “ifvDataCleaner”, a small helper program found in the “additional_tools” folder of the program files. - The proposed program predicts the likely location for glycosylation; however, it does not guarantee that this site will indeed undergo glycosylation. While the exposure of the side chain increases chances of potential glycan attachment, other factors also influence whether glycosylation will occur or not. Can authors comment on that? Several sentences describing other factors that contribute to sequon occupancy have been added to the discussion section of the manuscript. - Author did not consider oligomerization of protein in when calculation exposed potential glycosylation sites. Could it be a limitation of the program. Several sentences have been added in the manuscript to address the trimerization of the HA. - The definition of a sequon should be clearly stated. Given that not all glycosylation sites conform to the Asn-X-Ser/Thr rule, the specific amino acid sequences the program looks for are required. - Authors should provide a access to the program if possible. This has been done. The program is now accessible through GitHub and archived through Zenodo. Sincerely, Martin S. Zand MD PhD Attachments Attachment Submitted filename: Response.pdf https://doi.org/10.1371/journal.pone.0328174.r002
27 Jun 2025 Decision Letter - Cheorl-Ho Kim, Editor Future Sequon Finder - A novel approach for predicting future N-linked glycosylation sequon locations on viral surface proteins PONE-D-25-18727R1 Dear Dr. Zand, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Cheorl-Ho Kim, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr Zand, Thank you for your submission and revision. I would like to accept your revision, as I have previously requested to revise. Basically, it should be published and I am pleased to deal with your study. Thanks a lot Cheorl-Ho Kim Ph.D Editor Reviewers' comments: https://doi.org/10.1371/journal.pone.0328174.r003
Formally Accepted
Acceptance Letter - Cheorl-Ho Kim, Editor PONE-D-25-18727R1 PLOS ONE Dear Dr. Zand, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@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 Professor Cheorl-Ho Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0328174.r004

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