Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3

PLOS Pathogens

Dear Dr. Gaglia,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the points raised during the review process at Review Commons.

Please submit your revised manuscript by Jan 17 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Pinghui Feng

Academic Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

1) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

2) Please provide an Author Summary. This should appear in your manuscript between the Abstract (if applicable) and the Introduction, and should be 150-200 words long. The aim should be to make your findings accessible to a wide audience that includes both scientists and non-scientists. Sample summaries can be found on our website under Submission Guidelines:

https://journals.plos.org/plospathogens/s/submission-guidelines#loc-parts-of-a-submission

3) We noticed that you used the phrases 'data not shown' and 'unpublished data' in the manuscript. We do not allow these references, as the PLOS data access policy requires that all data be either published with the manuscript or made available in a publicly accessible database. Please amend the supplementary material to include the referenced data or remove the references.

4) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plospathogens/s/figures

5) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

- State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

- State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' Comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

Reproducibility:

?>

Dear Dr. Gaglia,

We are pleased to inform you that your manuscript 'Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3' has been provisionally accepted for publication in PLOS Pathogens.

As a personal note, I appreciate the significance of your findings described here as the two reviewers. I would like to point out that we previously reported that deamidation can dictate the gene-specific activation of RelA and IRF3, although conventional assays measuring RelA and IRF3 activation (e.g., phosphorylation and nuclear translocation) will not show apparent difference (Cell Metabolism, 2020; Immunity, 2025). These may provide some alternatives to explain the phenotypes your paper describes here.

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,

Pinghui Feng

Academic Editor

PLOS Pathogens

Blossom Damania

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 #1: The authors have been very responsive to reviewer concerns and nicely strengthened their main conclusions.

Reviewer #2: Main Finding: This study shows that the difference in IFN-β production during KSHV infection is determined by the levels and activation of host factors ATF2 and RelA, rather than IRF3. Using a reporter system, the authors demonstrate that even though IRF3 is activated in most infected cells, it is not the limiting factor. Instead, only a small subset of cells that have higher ATF2 protein levels and RelA phosphorylation are able to produce IFN-β.

Significance: This work explains the mechanism of why only a small percentage of cells produce type I interferon during viral infection. This uneven response is observed in many viral infections, but the reason was not well understood before. This study identifies ATF2 and RelA as the key factors underpinning this difference, which advances our understanding on how the innate immune system works at the single-cell level.

Comments:

1) Response to Reviewers: The authors answered all questions very well.

To address the concern about PAMP heterogeneity, they added Figure S3. This shows mitochondrial DNA is likely the ligand and proves that PAMP variation is not the main cause of the difference.

For the IRF3 activation markers, the authors added new data for IRF3 Ser396 phosphorylation (Fig 4F-G). This confirms that IRF3 activation is similar in both IFN-high and IFN-low cells, supporting the claim that IRF3 activation is not the bottleneck.

Also, the authors created a CMV promoter control line (Fig S2). This effectively rules out artifacts from random lentiviral integration.

2) Manuscript Structure: The structure of the manuscript is clear and the logic is strong. It starts by validating the reporter system, then systematically excludes stochastic noise and the TBK1-IRF3 pathway. At the end, it identifies ATF2 and RelA as the drivers of IFN induction. The addition of downstream target analysis (like ATF3 and TNFAIP3 in Fig S5D-E) further supports their conclusion.

3) Overall Assessment: The authors put a lot of effort into this revision. The new control experiments and biochemical data directly address the main concerns from the reviewers. The data is solid and the manuscript is now very complete.

**********

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

Reviewer #2: NA

**********

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: N/A

Reviewer #2: NA

**********

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: Yes: XINCHI XIE