Dear Dr/Associate professor wang,

Thank you very much for submitting your manuscript "Zebrafish mylipb attenuates antiviral innate immunity through two synergistic mechanisms targeting transcription factor 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. 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.

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,

Jacob S. Yount

Academic Editor

PLOS Pathogens

Sonja Best

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: These authors report zebrafish mylipb negatively regulates IRF3-mediated antiviral innate immunity through two synergistic mechanisms. 1) Mylipb promotes K6-linked polyubiquitination and autophagic degradation of IRF3. 2) Mylipb also acts as a decoy substrate for the phosphokinase tbk1 to attenuate irf3. Although the in vivo and in vitro experiments with overexpression and KO system convincingly support the view that zebrafish mylipb negatively regulates IFN-I signaling. However, the results that refer to the proof of regulatory mechanism are superficial (Fig 4, 5, 7), and some results are inconsistent. Furthermore the western blot experiments are conducted only with overexpression system.

Reviewer #2: In this manuscript, the authors report a role of mylipb in inhibiting irf3 functions in zebrafish. Using overexpression and under-expression strategies, the results suggest that mylipb inhibits virus-induced type-I IFN production via two synergistic mechanisms - induction of autophagic degradation of irf3 and inhibition of irf3 phosphorylation. mylipb interacts with irf3 and targets Lys352 for K6-linked polyubiquitination leading to autophagy-mediated degradation. Furthermore, mylipb gets phosphorylated by tbk1 and functions as a competitive inhibitor of irf3, which also works as a substrate for tbk1. Using this mechanism mylipb acts as a decoy substrate for tbk1, thereby attenuating phosphorylation of irf3 and cellular antiviral responses. Finally, they have demonstrated the physiological role of mylipb in vivo; knockout zebrafish are more resistant to SCVC infection due to increased viral replication and antiviral gene expression levels. Overall, there are several strengths in this study, including complementary mechanistic and physiological approaches and strong biochemical analyses. However, there are some weaknesses that need to be addressed.

**********

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: 1. It is necessary to study the phosphorylation, stability, ubiquitination of IRF3 in mylipb-KO cells, together with the proof of the endogenous IRF3.

2. From Fig 4G to 4K. The author proved that Mylipb induces autophagic degradation of IRF3 with WB and IF experiments. Several questions arise from this:

1) In fig 4K, the IF result indicated that overexpression of mylipb induce the LC3 puncta formation. LC3 puncta formation is a sign of autophagosome flux, indicating more LC3-I have been cleaved into the LC3-II. However, in fig 4J, the amount of lc3-II did not distinctly increase when overexpressing mylipb, indicating that mylipb did not induce the autophagosome flux. So the IF and IB results are inconsistent.

2) It is skeptical that the green signal of LC3 is undetected in the control group in fig 4K. In fig 4H, the bands of LC3-I and LC3-II between control group and mylipb-transfected group are without any difference. Whether the result is representative.

Reviewer #2: 1. The specificity of mylipb for irf3 is not solid. They should test if mylipb also targets irf7 and nf-kb proteins.

2. Inflammatory genes also contribute to the viral pathogenesis and induction of some of these genes should be tested in the whole fish study.

3. The species with slower mobilities in Fig 7 for irf3 and tbk1 were considered as phosphoproteins. However, some more specific studies, e.g., either phospho-specific antibodies or phosphatase should be used to come to a reasonable conclusion.

4. In Fig 5E, wt mylipb did not degrade irf3 protein, as expected. There was no explanation for this and also the mutant mylipb was used in this expt and for functional studies in Fig 6. mylipb mutant should be tested in the ubiquitination studies as well.

5. Autophagy-mediated degradation of irf3 has only been tested using inhibitors, which have off-target effects; genetic approaches (e.g., knockdown of autophagy components) should be used for validation.

**********

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. In fig 4A, the ISRE sequence is cloned from which species sample, because you have already labelled the IFN-luc with EPC or Dr. What are its sources?

2. In fig 5D, we can see that only mylipb-wt and 1-416 can induce the degradation of IRF3, while in fig 5B, the result is undetected in TCL.

3. For the ubiquitination assay result, three questions arise from this:

1) In fig 5C, IRF3 ubiquitination in △380-416 and C381S groups are still increased compared with the control group. So the result that E3 ubiquitin ligase activity was required for mylipb-mediated irf3 ubiquitination is skeptical. The ubiquitination assay of mylipb (1-280), (1-380), (1-416) should also conducted.

2) In fig 5F, the ubiquitination band is undetected in the K6R group, indicating that only K6-linked polyubiquitination chains can modify IRF3. However, in fig 5G, irf3 can be modified by any types of polyubiquitination chains. These results are contradictory.

3)It is suggested that 3-MA should appeared in ubiquitination assay results of fig 5.

4. In fig 7A, 7H, when transfected with TBK1, The HA-IRF3 has three bands, I want to know which band is unphosphorylated IRF3. If the three bands represent the whole protein level of IRF3, why both mylipb-wt and mylipb-C381S can induce the reduction of IRF3 protein level, which is contradictory with previous results.

5. In fig 7C, why the p-mylipb bands disappeared in TCL and IP sample.

6.The author declared that TBK1 phosphorylates mylipb. However, In fig 7D and E, the p-mylipb did not increase gradually with the overexpression of TBK1.

Reviewer #2: 1. In Fig 1, the levels of mylipb should be provided for the overexpression and knockout studies.

2. In Fig 2A, dose is not mentioned; also, the rationale for choosing 18h is not mentioned.

3. In Fig 3H-J the MOI of SCVC in EPC cell is missing.

4. In Figure 1B the titer of SCVC is mentioned but the MOI is missing.

5. In Figure 3D, line no 852, “After 24h, we infected (instead of transfected) the cells with SVCV”. The MOI is also not mentioned.

6. In Figure 6B and Figure 6D, MOI is missing.

**********

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 on PLOS Biology: http://www.plosbiology.org/article/info:doi%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/Associate professor wang,

We are pleased to inform you that your manuscript 'Zebrafish mylipb attenuates antiviral innate immunity through two synergistic mechanisms targeting transcription factor 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,

Jacob S. Yount

Academic Editor

PLOS Pathogens

Sonja Best

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):