Dear Professor Ma,

Thank you very much for submitting your manuscript "USP5 restricts anti-RNA viral innate immunity by deconjugating K48-linked unanchored and K63-linked anchored ubiquitin on IRF3" 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. There was clear enthusiasm for the work you presented. 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.

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.

Most of the reviewer requests are addressable. However, you will see that reviewer 3 requests the identification of the lysine residue in IRF3 that is modified with ubiquitin. This is a challenging experiment and not needed to support the claims, and thus this experiment is not required for resubmission. 

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,

Andrew Mehle

Academic Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

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: Here the authors note from published work that the deubiquitinase USP5 is downregulated during RNA virus infections. They confirm this finding in several RNA virus infections and examine whether this has functional consequences. They find that USP5 overexpression increases infections while its knockout promotes infection. They observe an inverse correlation between USP5 expression and IFNb induction, suggesting that USP5 negatively regulates interferon induction during infection. They narrow in on IRF3 by using IFNb promoter reporter assays with overexpression of RIG-I/MDA5 signaling pathway components. Direct interaction of USP5 and IRF3 that is increased by infection was confirmed with several independent assays. Mechanistically, they show that USP5 decreases K63- and K48-linked polyubiquitination of IRF3 and that inhibition of IFNb induction and increase of viral infection is dependent on USP5 catalytic activity. The data are clear and convincing in support of the conclusion that USP5 deubiquitinates IRF3 to suppress its IFN-induction activity. This finding is significant because mechanisms limiting IFN induction are important for preventing excessive inflammation and tissue damage. Aside from minor suggestions and one major issue with data interpretation/writing as noted in point 4 below, I support the publication of this manuscript.

Reviewer #2: The study by Qiao et al. entitled “USP5 restricts an-RNA viral innate immunity by deconjugating K48-linked unanchored and K63-linked anchored ubiquitin on IRF3” described a novel role of USP5 in regulating IRF3-dependent anti-RNA viral innate immunity. They have not only demonstrated a comprehensive regulatory mechanism that USP5 utilizes its dual-cleavage activity to deubiquitinate IRF3, but also provided a potential target for treating RNA virus infectious diseases. Overall, this is an interesting study with convincing results.

Reviewer #3: In this manuscript, the authors reported that the deubiquitinase USP5 is significantly downregulated during the host's innate immune response against RNA viruses, in an IFNAR-dependent manner. USP5 binds to IRF3 and efficiently removes both K63-linked anchored polyubiquitin chains and K48-linked unanchored polyubiquitin chains from IRF3. This action inhibits IRF3-driven transcription of IFN-β and the induction of IFN-stimulated genes (ISGs). The authors further demonstrated that USP5 negatively regulates IRF3-induced antiviral immune responses through its DUB activity. Overall, this is an interesting study with good novelty, which offers valuable insights into the regulation of USP5-mediated immune responses. However, several issues need to be addressed before consideration of publication.

**********

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: 1.The authors should add WB results for confirming the overexpression, knockdown, and knockout of USP5 in A549 cells and iBMMs. These results should be displayed adjacently to the corresponding functional experiments or included them in the supplementary figures.

2.In Figures S2A and S2B, the GFP fluorescence intensity is weak, and the differences is not so oblivious. It is recommended to extend the infection time or increase the viral infection MOI to enhance GFP intensity.

Reviewer #3: 1. Can the authors demonstrate which lysine residue of IRF3 undergoes deubiquitination by USP5?

2. Commercial antibodies for K48- and K63-linked polyubiquitination are available. It is good for the authors to detect endogenous IRF3 and its endogenous K48 and K63 ubiquitination using these antibodies?

**********

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: 1. The use of the word “restricts” in the title could be replaced by “inhibits” or “decreases.” The word restrict is usually used in this field in relation to restriction of virus infections (as opposed to restriction of innate immunity pathways that restrict virus infection as it is used here).

2. All bar graphs need to be re-made with symbols/colors that make more sense at first glance. The Key is confusing as open symbols are paired with solid bars and vice versa, making interpretation difficult.

3. The examination of STING KO cells in Fig 3 seems out of place and would not be a logical first place to start in terms of mechanistic examinations. This should be moved to later in the manuscript as a negative control or should be placed in the supplement.

4. Lines 180-190 describe the opposite of what the data show, and the conclusion at the end of this paragraph is the opposite of what they state in the previous lines. These are major mistakes in writing that are in opposition to the overall conclusions of the manuscript.

5. Line 192: This may be better written as “To explore roles of USP5 in RIG-I signaling, we assessed roles of…”

6. In Figure 5G, aren’t all of the ubiquitin chains shown in these blots expected to be anchored to IRF3 since it’s IP’d? It is thus confusing that the authors conclude that USP5 removes certain unanchored chains. Please clarify/explain further.

-Jacob Yount, The Ohio State University

Reviewer #2: 1.The manuscript contains several grammatical errors and types. The authors should check the manuscript carefully by themselves or use professional editing services for assistance.

2.In line 33, 'IFNAR' is mentioned for the first time, so its full name should be added.

3.In lines 149-151, to enhance readability, the text could be compressed by not repeating details and moving technical issues to the Materials and methods section or Figure legends.

4.Please provide the information in the methods section regarding the concentration of MG132.

5.Please discuss the potential role of unanchored ubiquitin in regulating host antiviral immunity against DNA viruses, since the cGAS-STING signaling pathway also requires IRF3 for induction of type I interferon.

Reviewer #3: 1.Figure 1F (left): the authors reported that viral infection can downregulate USP5 protein levels. However, in WT group, VSV upregulated Usp5 levels at time point 4 hrs. Please explain. In addition, total STAT1 levels were missing in Figure 1F.

2.Figure 2C, 2D: does USP5 overexpression or knockout affect virus titers?

3.Figure 3A-3C: does USP5 overexpression or knockout regulate basal expression levels of IFNB and ISGs?

4.Figure 3E: the p-STAT1 bands in Figure 3E are quite different from those in Figure 3D. Why? I would suggest to repeat Figure 3E to get clearer data.

5.Figure 4D: it is difficult to observe the staining of both USP5 and IRF3 in Figure 4D.

6.The authors reported that RNA viruses downregulate USP5 expression and that USP5 promotes RNA virus infection. How does USP5 affect DNA viruses?

7.In Figure 3F, the number of cells in each group varies considerably. Does USP5 regulate cell proliferation?

8.The description of results from lines 183 to 188 contains inaccuracies.

**********

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 Professor Ma,

We are pleased to inform you that your manuscript 'USP5 inhibits anti-RNA viral innate immunity by deconjugating K48-linked unanchored and K63-linked anchored ubiquitin on IRF3' 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,

Andrew Mehle

Academic Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

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 #2: The authors have addressed all my conerns. No further comments.

Reviewer #3: (No Response)

**********

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 #2: (No Response)

Reviewer #3: All concerns I raised have been well addressed.

**********

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 #2: (No Response)

Reviewer #3: (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 #2: No

Reviewer #3: Yes: Hui Zheng