Dear Dr Rajsbaum,

Thank you very much for submitting your manuscript "Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly" 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. Please note in particular the concerns of reviewer 2.

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,

Jens H. Kuhn

Associate Editor

PLOS Pathogens

Christopher Basler

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'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, the authors continue their research on the role of Ebola virus VP35 ubiquitination for viral replication. By intensively investigations of two VP35 mutants, carrying substitutions at amino acids 309 to arginine or glycine residue, K309R or K309G, the authors could show by applying reporter gene assays, interaction studies and recombinant viruses, that ubiquitination of VP35 K309 is essential for interaction with the viral polymerase L in order to initiate efficient viral RNA synthesis. In contrast, lack of VP35 ubiquitination leads to stronger interaction with NP, allowing nucleocapsid assembly and packaging into progeny virions. All together, these results suggest ubiquitination of Ebola virus VP35 plays a key role in switching the two different functions of VP35 in transcription vs nucleocapsid assembly.

The manuscript is very well written and provides new evidence for the mechanistic role of VP35 ubiquitination during viral infection by modulating the two distinct function of VP35 in transcription and nucleocapsid assembly through ubiquitination of VP35 K309. The experiments are solidly executed and are very intensively controlled.

There are only minor issues to be addressed

Reviewer #2: This manuscript by van Tol et al., explores the role of Ebola VP35 ubiquitination. This work builds on a previous observation that Ebola VP35 is ubiquitinated on a specific basic residue, K309. The authors extend this initial observation by generating recombinant viruses with mutations at K309 (K209G and K309R) in an attempt to define the molecular mechanism. Much of the data in the manuscript support the original observation from the same group, Bharaj et al. 2017, with the major contribution, albeit not entirely surprising is the fact that mutations within the C-terminal RNA binding interferon inhibitory domain (RBD/IID) does not impact its interferon inhibitory function. This is consistent with multiple prior reports which suggested that K309 plays a minor role in the central basic patch region. Authors also generated recombinant viruses and this was important and has the potential to impact our knowledge. While the role of posttranslational modifications, including ubiquitination are likely to impact Ebola replication, the data in the manuscript overall is not rigorous. Often the error bars/statistical considerations are missing and the data/replicates are missing. Therefore, while the manuscript presents and important idea, the significance and the impact of the data lacks an ability to support the hypothesis. For example data in Figure 3 shows a log difference in panel A, but a black and white difference in panel C at MOI of 5 a dn 48 hours time. Panel C is using a normalization to actin. Using multiple Y-axis, the authors are either trying to convince themselves and the reader or enhance a modest effect. Unclear which of this is true. Finally, data in Figure 6 is intriguing. If the ubiquitination was truly the reason for why VP35 does not bind to Ebola viral L, then using WT VP35 and WT L1-505, one should see a disruption in TRIM6 KO cells. That data is missing. Again, in multiple instances, the authors do not provide rigorous data to support their ideas. For these reasons, there is promise, but does not deliver with respect to biology.

Reviewer #3: This is a thoroughly designed and executed study demonstrating the significance of ubiquitination of EBOV VP35 at lysine 309 in viral transcription and assembly. The authors generated recombinant K309R and K309G EBOV mutants to differentiate the role of ubiquitination and the basic property of the K309 residue in regulating virus infection. It appears that Lysine ubiquitination is not essential for VP35 to antagonize IFN-1 activity but was necessary for transcription and proper virus assembly. Several approaches have been used to drive the main conclusions. The data is also supported by rigorous statistical analysis.

The rescue of the mutant viruses and demonstration of their attenuating phenotype in vitro assays by itself is very promising for developing countermeasures. This is further enhanced by detailed molecular studies into the mechanism of action.

**********

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: There are only minor issues to be addressed

Reviewer #2: For example data in Figure 3 shows a log difference in panel A, but a black and white difference in panel C at MOI of 5 a dn 48 hours time. Panel C is using a normalization to actin. Using multiple Y-axis, the authors are either trying to convince themselves and the reader or enhance a modest effect. Unclear which of this is true. Finally, data in Figure 6 is intriguing. If the ubiquitination was truly the reason for why VP35 does not bind to Ebola viral L, then using WT VP35 and WT L1-505, one should see a disruption in TRIM6 KO cells. That data is missing.

Reviewer #3: I have one concern that needs clarification.

Fig. 6E. This is a very nice experiment; however interpretation of the results didn’t seem right due to the following reasons:

1) It appears that OTU co-expression with VP35 or VP35-K309R was sufficient to enhance VP35-NP interaction. This appears to be the case at both low and high expression of VP35. However, the corresponding densitometry analysis didn’t correlate with the Western blot data. Based on Western blot alone, it appears that there is some ubiquitin dependent component that was presumable destroyed by OTU that enhanced NP-VP35 interaction. This is in addition to the enhancement of NP-VP35 due to OTU mediated loss of ubiquitin modification at K309.

2) Can the authors demonstrate, as a control, the level of expression of the Ubiquitinated-VP35 species in the lysates of mock, OTU and 2A transfected cells. This will also check if there are any additional residues on VP35 (other than K309) that are ubiquitinated (like in Fig. 1c). It is possible that these additional residues are influencing VP35-NP interaction. However, it is understandable if the authors find such a demonstration to be technically challenging. But additional insights into this interesting observation should be provided.

In general the data just going by the Western blot is interesting but the interpretation was confusing and needs clarification to make the claim that NP-VP35 interaction is dependent only on K309 ubiquitination

**********

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: Minor comments.

1. Line 83: the abbreviation for antigenomic RNA, agRNA, is unusual. Conventionally, the terms and abbreviations for vRNA (genomic RNA) and cRNA (copy RNA, antigenome) are more widely used.

2. Line 175: To this reviewer, usage of A549 cells, a lung cell line, for Ebola virus infection seems unusual. There are more widely used IFN competent cells like Huh7 cells, a hepatoma cell line, liver cells are also infected during an authentic viral infections. Could the authors please comment on their rational to use the A549 cells?

3. Line 190 and line 193 Typo Ifnb. Must be IFNß. Isg must be ISG

4. Fig. 4A. To me, it is not clear why the authors also assed reporter gene activity at lower doses of VP35 and mutants. The ratio between VP35 and NP in the minigenome assay setting (Hoenen et al., 2004, Wenigenrath et al., 2010) is very critical for efficient transcirpiotnal activity. Altering this ratio by just transfecting a quarter of the usual dose of VP35 will of course affect transcriptional activity. However, this must not have anything to do with the VP35 Ub mutations at all.

5. Fig. 6C. Expression control of L is missing.

6. Fig. 6F. A quantification of Western Blots is missing. While the authors state that interaction of mutants with VP30 is not impaired (line 346), the blot shows another picture. Also with respect to Fig 7A, where K309G infection shows reduced VP30 incorporatio, it is highly recommended to include here a statistical analysis of more than 1 Blot to interprete these results more precisely.

7. Fig. 8. It is highly recommended to replace the reddish, pinkish colours with different types to improve clarity of the model. Also the particles that are “produced” are really hard to recognize instinctively.

Reviewer #2: statistics/repeats, use of acceptable Y-axis and describe rationale when unusual nationalizations are done.

Reviewer #3: No issues in this section

**********

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

Reviewer #3: 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 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 Rajsbaum,

We are pleased to inform you that your manuscript 'Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly' 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,

Jens H. Kuhn

Associate Editor

PLOS Pathogens

Christopher Basler

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):

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 present a thouroughly revised manuscript where they also included important new experiments that support there hypothesis in the role of VP35 K309 Ub. They also revised there statistical analysis.

**********

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: n.a.

**********

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: All minor issues were covered.

**********

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