Dear Dr. Rosenthal,

Thank you very much for submitting your manuscript "Structural and functional characterization of the Sin Nombre virus L protein" 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.

The reviewers and I appreciate the significance of this work. The reviewers made constructive comments most notably on the biochemistry as it relates to protein purity, and to activities. I agree with those comments. As addressing some of those comments may require additional experimentation the present recommendation is major revision.

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,

Sean P.J. Whelan

Academic Editor

PLOS Pathogens

Matthias Schnell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

The reviewers and I appreciate the significance of this work. The reviewers made constructive comments most notably on the biochemistry as it relates to protein purity, and to activities. I agree with those comments. As addressing some of those comments may require additional experimentation the present recommendation is major revision.

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 this manuscript, Meier and colleagues establish an expression and purification protocol for the L protein with a K124A endonuclease mutation of Sin Nombre virus (SNV). They perform functional characterisation of the L protein using RNA-binding, endonuclease and polymerase activity assays. In the second part of the manuscript, they present a 3D model of the L protein core region containing the RdRp domain, in complex with 5’ promoter RNA, obtained using single-particle cryo-EM. The authors compare their biochemical and structural observations with those for other segmented negative-sense RNA viruses (sNSVs).

Overall, this is an important addition to a growing body of literature on the structure and function of L proteins of sNSVs. The study highlights some interesting specificities of hantavirus L proteins such as the presence of the Hanta and California-like insertions in the palm domain of the RdRp. Generally, the manuscript is well-written and the data are clearly presented; for most parts the data support the conclusions but there are some issues that will need to be addressed.

Reviewer #2: This manuscript by Meier and coworkers examines the L protein polymerase of Sin Nombre hantavirus. Purification of the Sin Nombre virus L protein has proven problematic due to its highly active endonuclease activity, which suppresses protein expression. In this manuscript, the authors describe how they successfully purified the L protein by introducing a mutation into the endonuclease domain. The purified protein was analyzed in a series of biochemical assays that examined its endonuclease and RNA synthesis activities and the structure of the Sin Nombre virus L protein in association with RNA was determined. The manuscript adds to the growing body of literature regarding segmented negative strand RNA virus polymerases and provides valuable information that can be leveraged for future structure-informed functional studies. While the manuscript adds valuable new information to the field, some of the results from biochemical assays raised some questions that should be addressed.

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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: Fig. 2. I find it surprising that the radiolabelled RNA is almost fully degraded but there is no obvious depletion of the fluorescently labelled RNAs even at the highest metal concentrations. These data would be consistent with a phosphatase contamination in the L protein prep (could the authors include an SDS-PAGE of the L protein prep used?). The authors should check that the “endonuclease” activity they observe is sensitive to DPBA (as in the subsequent figure). The data in Fig. S3. (not referred to in the manuscript) suggest that the observed “endonuclease” activity is specific to polyA but the significance of this is not discussed. Given that all these data were obtained with an endonuclease mutant I wonder about the significance of these data; they seem to add little to an otherwise strong manuscript.

Fig. 3. In the in vitro polymerase activity assay the authors use an 18 long template but observe mostly longer and heterogeneous products that are unlikely to represent authentic transcription products. This issue is addressed in the Discussion and in fact the authors present a more convincing result using a 3’ phosphorylated RNA template (Fig. S4) where a major 18 nt product is observed with some longer products that can be explained by template switching. I wonder why the authors don’t show this result in the Results section instead of data in Fig. 3 that are difficult to interpret. A further issue with the data in Fig. 3B is that the DPBA experiment lacks a positive control; also, DPBA seems to change the band pattern of products in the Mg2+ conditions but this is not commented on. Comparing band patterns in panel A and B I wonder whether the label for Mn2+ and Mg2+ is switched around. Panel A lacks size markers.

Reviewer #2: 1. It would be helpful to show the purified polymerase on a Coomassie stained denaturing polyacrylamide gel to provide an indication of polymerase purity.

2. Fig. 2: Panel A does not appear to be consistent with panel B, lanes 1-5. In panel A, 40 mer poly A was efficiently cleaved by the endonuclease, whereas in B a 27 mer poly A showed minimal cleavage. It should be determined if this is due to the RNA length or the presence of the fluorescent tags.

3. It is possible that the degradation observed in Fig. 2 is due to a contaminating RNAse given that the activity is not ablated by the K124A substitution. Are there additional mutations that could be introduced to ascertain if the endonuclease activity that is observed is due to the SNV L protein? Alternatively, if DBPA has specificity for viral endonucleases perhaps this could be used to confirm the specificity of this cleavage.

4. Fig. 3: It appears that the data presented in Fig. 3A and Fig. 3B are inconsistent with each other. In panel A, 10 mM MnCl2 appears to completely inhibit RNA synthesis activity, whereas this is not the case in Fig. 3B. If this is because the panel B was exposed for much longer than panel A, this should be stated in the results section or legend. If there is another reason, an explanation should be provided.

5. Fig. 3B: The pattern of RNA product bands is very different in the reactions that contained MgCl2 +/- DBPA. An explanation should be provided for these results.

6. Fig. 3 and subsequent RNA gels, there are products as long as 40 nt in length, although the template is only 18 nt. In the discussion section (lines 518-522) it is suggested that this is due to template switching. However, in Fig. 5C, there were bands of ~15 and 18 nt, even in reactions lacking GTP. It is difficult to conceive how the polymerase generates products of 15 and 18 nt in length by template switching in -GTP conditions. Is it possible that the RNA is not fully denatured? In addition, the discrepancy between the template and product length should be mentioned in the Results section.

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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: Fig. 1 and line 143. These data are suggestive that the interaction of the L protein with the 5’ RNA is stronger than with the 3’ RNA but no affinity measurements were performed to be able to conclude that the interaction is “significantly stronger”. Please tone down the language.

Fig. 4. Size markers are lacking.

Line 322. Fig. S1A is referred to here; is this correct?

Fig. 5. The authors suggest that there is a single binding site for the di/tri nucleotides (template sequence at the bottom of panel A). Due to the repetition of the AUC motif at the 3’ end these primers can all bind at multiple sites in the template; should this be considered?

Fig. 8 and Fig. S7. These two figures could be joined as a single supplementary figure and perhaps discussed in the Discussion rather than being presented as Results.

Fig. S1A. The legend refers to 3’ RNA but 5’ RNA is shown in the figure.

Fig. S5C. The authors indicate only one class contains RNA. It would be informative to include particle numbers for these classes to give an idea of RNA occupancy.

Fig. S6. Include nt labels to orient the reader.

Table S1 and line 668. The table states about 500k particles were used in the final reconstruction; however in the methods 90,361 are mentioned. Please check.

Line 528. “extent”

Line 589. Presumably “300 mM NaCl”.

Lines 595 and 598. No “reaction” occurs in an electrophoretic mobility shift assay.

Line 625. Please check that 0.5 mM GTP is correct; this would result in a very low ratio of 32P-GTP most likely not sufficient for detecting the RNAs.

Reviewer #2: 7. Line 299-300 – “ Taken together, our data suggest that the SNV L RdRp has a higher specificity for purines, indicative of a low RdRp fidelity”. This is worded oddly. It would make more sense if it was written as “Taken together, our data suggest that the SNV L RdRp has a low specificity for pyrimidines, indicative of a low RdRp fidelity”.

8. Line 475: the inhibitory effect of the 5´ RNA was reduced in the presence of short primers. Could this be because the primer competed with 5´ RNA for binding to the 3´ RNA? Is the sequence of SNV different than for other viruses, do the 5´ and 3´ RNAs have a higher Tm?

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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.

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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

Dear Dr. Rosenthal,

Thank you very much for submitting your manuscript "Structural and functional characterization of the Sin Nombre virus L protein" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The manuscript is in principle accepted but please respond to the minor comment of reviewer 2.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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.

Thank you again for your submission to our journal. 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,

Sean P.J. Whelan

Academic Editor

PLOS Pathogens

Matthias Schnell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

The manuscript is in principle accepted but please respond to the minor comment of reviewer 2.

Reviewer Comments (if any, and for reference):

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 authors have revised their paper satisfactorily addressing all the reviewers' comments. I have no further comments.

Reviewer #2: The reviewers' comments have been addressed and the conclusions appear to be sound. The only query I have is that on lines 210-213 it is stated that "we also detected degradation of the Cy5-labeled viral 3' and 5' RNAs 1-18 in substoichiometric RNA concentrations relative to the L protein, albeit with lower efficiency than the poly A substrate". However, looking at panel 2B, it is difficult discern a difference in cleavage of the polyA RNA versus the 3' 1-18 or 5' 1-18 RNAs. Revision of the accompanying text or an explanation would be helpful.

**********

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: (No Response)

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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: 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

References:

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.

Dear Dr. Rosenthal,

We are pleased to inform you that your manuscript 'Structural and functional characterization of the Sin Nombre virus L protein' 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,

Sean P.J. Whelan

Academic Editor

PLOS Pathogens

Matthias Schnell

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):