Dear Professor Chen,

Thank you very much for submitting your manuscript "ATG7/GAPLINC/IRF3 axis plays a critical role in regulating pathogenesis of influenza A virus" 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 that new experiments, new results and extensive text revisions are required to address reviewer comments. This includes experiments addressing mechanisms by which ATG7-GAPLINC reduce IRF3 phosphorylation, tests for effects of ATG genes beyond ATG7 in controlling IAV, and additional control tests involving CRISPR KO and complementation assays that further determine credibility of findings. Text needs to be significantly revised in ways that reveal clear premises and logical progressions of the work as it builds a case for the ATG7-GAPLINC-IRF3 axis in IAV infection.

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.

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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,

Tom Gallagher

Guest 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

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Please note that new experiments, new results and extensive text revisions are required to address reviewer comments. This includes experiments addressing mechanisms by which ATG7-GAPLINC reduce IRF3 phosphorylation, tests for effects of ATG genes beyond ATG7 in controlling IAV, and additional control tests involving CRISPR KO and complementation assays that further determine credibility of findings. Text needs to be significantly revised in ways that reveal clear premises and logical progressions of the work as it builds a case for the ATG7-GAPLINC-IRF3 axis in IAV infection.

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 study by Chen et al., the authors investigate the role of autophagy protein ATG7 on influenza A virus (IAV) replication and cell autonomous immune responses. The authors report that shRNA knockdown of ATG7 in the human lung epithelial cell line A549 decreased IAV replication. In contrast, overexpression (OE) of ATG7 in A549 cells increased IAV replication, indicating that ATG7 promotes IAV replication. These findings are further supported by in vivo studies in a tamoxifen inducible Atg7 ko mouse model, where loss of Atg7 decreased IAV viral titer in the lungs. Through RNASeq analysis, the authors identified a non-coding RNA GAPLINC that was lower in IAV infected ATG7 KD cells. shRNA KD or OE of GAPLINC decreased or increased IAV replication, respectively. Interestingly, OE of GAPLINC in ATG7 KD cells was sufficient to rescue IAV replication, indicating that GAPLINC acts downstream of ATG7 and that the lowered IAV replication in ATG7 KD cells is due to decreased GAPLINC levels. Next, the authors investigated if ATG7 and GAPLINC promotes replication by reducing RLR signaling and report that ATG7 suppresses the phosphorylation of IRF3, thereby promoting IAV replication. Similarly, the non-coding RNA GAPLINC reduced the levels of p-IRF3. The authors conclude that ATG7 and GAPLINC promote IAV replication by suppressing IRF3 activation. Although these are interesting findings, the study lacks mechanistic insights into how GAPLINC suppresses IRF3 activation. In addition, the study relies on observations with ATG7 KD/ Atg7 KOs, which needs to be supported by KD or KO of other autophagy genes. Furthermore, some of the RLR suppression studies need to be validated with CRISPR KO or complemented KD cells with shRNA resistant cDNA of ATG7 and GAPLINC.

Reviewer #2: Biao Chen et., al demonstrate ATG7, an essential autophagy effector enzyme, suppresses IRF3 activation and interferon production via lncRNA GAPLINC, revealing an autophagy-independent mechanism whereby ATG7 restrains host innate immunity and unveiling a critical role of ATG7/GAPLINC/IRF3 axis in regulating IAV pathogenesis. These results show that ATG7 has multiple biological roles beyond autophagy, and provide an important insight into the complicated interplay between host and IAV. In general, there are many points involved in the manuscript with large workload and the experimental results are clear and the evidence is relatively sufficient.

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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: Major concerns:

1. The lack of mechanistic insights into how ATG7 and GAPLINC suppress IRF3 activation. The role of autophagy in RLR signaling and GAPLINC in the suppression of inflammatory gene expression has been reported by others. I think further investigation of the underlying mechanisms will strengthen the manuscript.

2. It is unclear how the expression of GAPLINC is lowered by loss of ATG7.

3. It needs to be demonstrated if there are changes in ATG7 and GAPLINC during IAV infection and how this impacts the overall IAV life cycle.

4. Are the levels of Gaplinc lower in Atg7 KO mouse lungs? The findings will be strengthened if the authors could demonstrate their findings from A549 cells in mouse Atg7 ko cells.

5. The data for autophagy independent role of ATG7 is weak. The majority of the data relies on chemical inhibitors, which can also block IAV infection by blocking endosomal acidification (HCQ).

Reviewer #2: Major comments:

The logic of the article is a bit confusing and needs to be revised. For example, the authors found that ATG7 could promote IAV infection in an autophagy-independent manner, and then found and explained that lncRNA GAPLINC was downregulated in ATG7 knockdown A549 cells immediately. Later, the authors directly jumped to the context of innate immunity response by ATG7 and guess lncRNA GAPLINC may play critical roles in it. Finally, the relationship between these three was linked together by all the authors’ suppose instead of experimental results. As a result, the research content does not demonstrate a hierarchical and closely connected logic.

Actually, when the authors found that ATG7 could promote IAV infection in an autophagy-independent manner and in order to clarify this molecular mechanism, RNA-Seq experiments were conducted. Compared with RNA-seq results of ATG7 wild-type and knockdown cells, finding that interferon type I and III were upregulated in ATG7 knockdown cells, indicating that ATG7 may regulate IAV infection by influencing innate immune response. Then, experiments were conducted to demonstrate that ATG7 could assist in IAV infection by inhibiting the phosphorylation of IRF3. Next, how does ATG7 regulate this process? Then according to RNA-seq results and it came to the relevant content of lncRNA GAPLINC. This kind of logic is more compact and rigorous.

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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: Minor concerns:

1. Most of the data presented in the manuscript does not indicate the time points for analysis (hpi or dpi?)

2. For hemagglutination titers, the y-axis label should say HA titer and not viral titers.

3. There is a two-fold difference in viral titers. Surprisingly, the NP levels on the western blot are so different.

4. Nomenclature for mouse gene names are incorrect.

5. It will be informative for the readers to show the individual mouse lung titers as a scatter plot instead of a bar graph.

6. Timeline between Tamoxifen injection and viral infection is not clear.

7. Some of the graphs are overlapping and the legends/numbering are blocked.

8. GAPLINC- RNASeq data doesn’t include mock controls. It is important to include this data as prior studies indicate that KD of GAPLINC increases the basal expression levels of inflammatory cytokines.

Reviewer #2: Minor comments:

1. S1A Fig showed that A549 cells expressing ATG7 shRNAs could still detect ATG7 protein, but in Fig 3G or S3B Fig, A549 cells expressing ATG7 shRNAs seemed appeared no ATG7, just like ATG7 knockout cells. It needs to be modified and explained.

2. In Fig 2A, authors detected ATG7 protein levels in ATG7 flox/flox/UBC-CreERT2 mice tissues, but instead of thymus, all the tissues showed a high level of ATG7 in ATG7-/- mice. I don't know whether “ ATG7 knockout mice” described in the article are suitable.

3. In the context of effects of ATG7 on the IRF3 activation, authors only checked IRF3 phosphorylation. What’s the effects of ATG7 on IRF3 dimerization and nuclear translocation. Authors should add these results to make the article conclusion stronger.

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Reviewer #1: No

Reviewer #2: No

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Dear Professor Chen,

We are pleased to inform you that your manuscript 'ATG7/GAPLINC/IRF3 axis plays a critical role in regulating pathogenesis of influenza A virus' 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,

Tom Gallagher

Guest 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):

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 addressed all of my previous concerns.

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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: (No Response)

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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: (No Response)

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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