PONE-D-24-53270Human cytomegalovirus infection induces L1 expression through UL38-dependent mTOR-KAP1 pathwayPLOS ONE

Dear Dr.  Ahn,

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PONE-D-24-53270R1Human cytomegalovirus infection induces L1 expression through UL38-dependent mTOR-KAP1 pathwayPLOS ONE

Dear Dr. Ahn,

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

==============================

ACADEMIC EDITOR:  I ask the authors to respond and address the reviewer 4 comments

==============================

Please submit your revised manuscript by Mar 01 2025 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 plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• 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, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Gianmarco Ferrara, PhD, MVD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I did not see their responses to the reviewers. I did not see their responses to the reviewers. I did not see their responses to the reviewers.

Reviewer #2: The research reveals that HCMV UL38 serves as a key viral regulator of L1 expression by activating the mTOR-KAP1 pathway. This interaction demonstrates a cooperative relationship between HCMV and L1, with HCMV leveraging L1 to boost its replication. Unraveling this mechanism sheds light on how HCMV manipulates host cellular functions and its possible effects on genomic stability and disease progression. However, the study primarily focused on the UL38 protein, potentially overlooking other viral or host factors involved in L1 activation. Additionally. the article would be enhanced by expanding the discussion to include the broader implications of the mTOR-KAP1 pathway, such as its involvement in other viral infections or diseases.

Reviewer #3: In this study Park and Ahn demonstrated that through the HCMV viral protein UL38 activates mTOR that phosphorylates KAP1, reducing its epigenetic repression and leading to increased chromatin accessibility of L1 promoter region. The study well conducted and well written and contributes to the understanding of the mechanism by which HCMV leads to expression of the LINE-1 retrotransposon. I believe that the manuscript has original data significant to the field and clarity of presentation and is suitable for publication in this current form.

Reviewer #4: The authors present compelling evidence that KAP1 phosphorylation downstream of mTOR signaling induces expression of the ORF1 protein of L1 retrotransposon, and that HCMV UL38 is required for this induction. While the data are convincing, most of the protein expression data are not quantitated and it is not clear if experiments were performed more than once, making it difficult to accept the conclusions drawn by the authors. Furthermore, the study could be strengthened by additional experiments to determine whether HCMV UL38 is sufficient to induce expression of L1 and whether these effects have implications for viral replication as has been previously reported for L1. Additionally, the authors should take care to more clearly explain how data was quantified as the results section and figure legends do not provide sufficient detail. Lastly, the authors at times draw conclusions that are not directly supported by the data and so should amend their statements to more accurately reflect their results. Major and minor concerns are listed below.

Major concerns:

1. Western Blots are not quantified, and it is not clear if each Western blot was performed more than once. The authors draw conclusions about changes (or lack of changes) in protein expression, but this should be quantified for the blots shown and a graph should be made from at least three replicate experiments. For an example, the authors state that total KAP1 levels “did not decrease” as shown in Fig 2B, but the levels appear to increase. This should be quantified and discussed in the revised manuscript. Quantification of blots needs to be done for Fig 1A-B, 2A-C, 3A-B, 4D.

2. Lines 150-152: “We found that L1 ORF1 protein expression increased throughout the 72-hour HCMV life cycle indicating that L1 expression was upregulated and L1 ORF1 accumulated over the course of HCMV infection (Fig 1A).” At what time points do you observe increased L1 expression? It does not appear to increase at 24hr, but this is not quantified. Is the upregulation at later time points indicative of when during the viral life cycle L1 is induced? Does this correlate with UL38 expression? Is it dependent on viral gene expression, DNA synthesis, etc? These points should be discussed in the manuscript.

3. Fig 1B. and 3A. Authors state that UV-HCMV does not induce L1 expression or phospho/total S6K, respectively, but there appears to be a slight induction. This should be quantified and discussed in the revised manuscript.

4. Your data is clear that UL38 expression is necessary for driving L1 expression, but is UL38 sufficient to increase mTOR signaling and induction of L1 expression? This could be tested using a plasmid expressing UL38. Or are other HCMV factors required to stimulate L1 expression?

5. Previous studies by this group showed that L1 expression during HCMV infection is important for viral replication. This would suggest that a UL38 mutant, which results in reduced L1 levels during infection, would exhibit a growth defect. The authors should provide growth curves for the UL38 mutant virus. Additionally, the authors should provide more discussion about how the findings in this study fit in with the data previously published for L1 expression during HCMV infection.

Minor concerns:

1. Fig 1D-E. Is the data RLU or is it relative to non-infected cells (set to 1)? Either is fine, but please describe this in more detail in the figure legend.

2. The figure legend for Fig 1F refers to up/down panels, but it should be left/right.

3. Also, for the Fig 1F legend please describe how the data is shown. It is unclear what “relative fold enrichment” means when the results state that the calculated shift in Cq values between nuclease-treated and non-treated samples was calculated.

4. Lines 56-58 and 334-335 are missing references.

5. One lines 205-208, the %knockdown and %increase in expression are not consistent with the data shown in Fig 2A.

6. Lines 216-218: “upon HCMV infection, viral factors inactivate...”: you have not thus far shown that it is a viral factor, just infection itself (but not likely entry since you don’t observe changes with UV-HCMV). This could be indirect effects. Please change the statement to something that more accurately reflects the data. Either HCMV infection induces, or HCMV viral gene expression induces… Your data suggests it is at later time points (48-72 hr), suggesting that it is not IE gene expression.

7. Fig 2A. legend does not fully explain values quantified. Were signal intensities quantified by ImageJ and normalized to a loading control?

8. On lines 267-269, it is stated that HCMV entry was unaffected. This is not exactly what the data show. The data shows equivalent levels of viral DNA, suggesting that there was no defect in entry, viral gene expression, etc. but the authors did not perform an entry assay. Please more accurately describe the results.

9. For Fig 4B-C, I cannot determine exactly what was measured based on the figure legend (what UL38 expression is normalized to, how viral DNA was measured). Also, the legend says that 4C shows UL38 expression when it does not. Please make these additions and changes.

10. In the discussion, there are multiple mentions of “the HCMV factor”. I’m assuming UL38, but this should be stated more clearly.

11. On lines 323-324, it is stated that, “the HCMV factor induces epigenetic derepression by interacting with cellular epigenetic regulator KAP1…” The data does not show a direct interaction, so this statement should be amended to reflect the data.

12. On lines 230 and 232, it should read, “the mTOR pathway” and not “mTOR pathway”. Also, on line 239, it should read “KAP1” and not “the KAP1”.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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[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.]

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. Registration is free. 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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Human cytomegalovirus infection induces L1 expression through UL38-dependent mTOR-KAP1 pathway

PONE-D-24-53270R2

Dear Dr. Kwangseog Ahn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gianmarco Ferrara, PhD, MVD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: No

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

Reviewer #4: The authors responded to all of my comments and also provided additional data to address my concerns.

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7. 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 #3: No

Reviewer #4: No

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