Dear Dr. Grimes,

Thank you very much for submitting your manuscript "Structural characterisation of the Hantaan virus polymerase describes a full-length polymerase conformation and reveals large rearrangements of the polymerase active site." 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 at least one independent reviewer. In your particular case,  we sought for multiple independent reviews but only manage to get one expert review. Instead of holding off on a  decision, we decided to proceed with the one review and a close look by the Section Editor.  In light of the review (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewer's comments.

Pleas pay particular attention to how the novel features interpreted in this study can be determined by the resolution of cryoEM amps obtained. 

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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,

Benhur Lee

Section Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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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: The manuscript by Keown et al. describes several structures of the HTNV polymerase including protein constructs of two different lengths and with and without bound RNA. While significant structural work on HTNV polymerase has been previously published, this work details several new findings including conformations of motif F that block template binding and a full-length structure of the polymerase. Despite enthusiasm for these novelties, there are several concerns primarily relating to the interpretation of the authors’ EM maps. While the findings are interesting both the content and the descriptions in the text should be rigorously reevaluated.

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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: Line 260: The identification of a nucleotide in the active site is unjustified. Even taking the map threshold very low, it is not possible to distinguish any part of the nucleotide or magnesium ions from the noise. It isn’t clear how the authors produced figure S6A, but I cannot reproduce it with their maps.

Fig S7: Given the weak nature of the bands and large number of alternate products. I would prefer to see replicates of the ApG extensions with polymerase mutants.

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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: Line 133: Claims of an ordered endonuclease domain in 8P1K seem overstated. The resolution of this domain is significantly lower than the core portion of the structure and much of the domain is entirely absent at low threshold.

Line 137: Not clear what the reference to “261-273” is referring to as it appears to not be the endonuclease, core lobe or linker.

Line 158: the observation of insertions in the palm domain appear to make a claim to novelty, but these structures were previously observed in Durieux et al. 2023.

Line 189: Not clear what “different” piece of 5’ vRNA the authors are referring to here. 8QH3 appears to have an identical RNA sequence bound at this position.

Line 218: The C-terminal domain, while clearly present in the map, is largely disordered. While the domain as modeled is one possible interpretation of this map, structural homology searches and comparisons mentioned in the text are somewhat dubious.

Line 229: While there is clearly a shift in the endonuclease domain between 8P1K and 8P1L, the poor density for the endonuclease in map 17352 precludes any sort of conclusion about changes in this regions conformation or contacts as seen in the full-length structure.

Line 232: The authors have actually built three conformations for this loop. The density for this region in 8P1M/17354 is poor though and it isn’t sure what the justification for an alternate build here is.

Line 310: Presumably “the position” of motif E being referred to is the alpha helical conformation?

Line 325: I am not convinced that the authors are seeing four distinct conformations of motif E. 8P1J, 8P1K and 8P1M all have the same short helix, 8P1L contains the longer helix and 8P1N contains the beta-sheet. It isn’t clear here what the authors are referring to as the “ordered loop” arrangement.

Line 570: Update to indicate 8P1L pairing with EMDB-17353. 8P1M appears to be 5’ vRNA + ATP and 8P1N appears to bound to just 5’ vRNA. Database entries appear to be correct.

Fig 3A, 4ACD: Please avoid using yellow text, it is very hard to read.

Fig S2, S4: Figures and text need to be at a size that they can be read

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Reviewer #1: No

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PPATHOGENS-D-24-00803R1Structural Characterization of the Full-length Hantaan Virus PolymerasePLOS Pathogens Dear Dr. Grimes, 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 submit your revised manuscript within 60 days Jan 19 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 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, Matthias Johannes Schnell, PhDSection EditorPLOS Pathogens Benhur LeeSection EditorPLOS Pathogens Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064  Journal Requirements:

Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

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.

 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.] 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. Grimes,

We are pleased to inform you that your manuscript 'Structural Characterization of the Full-length Hantaan Virus Polymerase' 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,

Matthias Johannes Schnell, PhD

Section Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):