Integrative RNA-seq and ChIP-seq analysis unveils metabolic regulation as a conserved antiviral mechanism of chicken p53

ABSTRACT The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53’s antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53’s genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens. IMPORTANCE The current study conducted a comprehensive analysis, comparing molecular networks underlying chp53’s antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

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Sincerely, Manjula Kalia Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): In the article by Lu Cui and colleagues, the author investigates the molecular networks related to chp53's antiviral activity against IBDV, ALV-J, and ILTV.ChIP-qPCR results indicate that chp53 regulates host cellular metabolism across various viruses.
In the article by Lu Cui and colleagues, the author investigates the molecular networks related to chp53's antiviral activity against IBDV, ALV-J, and ILTV.ChIP-qPCR results indicate that chp53 regulates host cellular metabolism across various viruses.
The following are comments on manuscript 1. Please incorporate the multiplicity of infection (MOI) of viruses in the materials and methods section.
2. Figure 7: Please clarify why only metabolic genes were chosen for validation.

*Accepted
The manuscript by Lu et al. presents a comprehensive study on the antiviral role of p53, a well-established antiviral factor in numerous human and animal viruses, including chicken viruses.The authors have performed an integrative analysis of RNA-seq and ChIP-seq data across three major chicken pathogens: IBDV, ALV-J, and ILTV.Their findings suggest that targeting the virus-host metabolic interaction through the regulation of p53 could serve as a universal strategy for antiviral therapies in chickens.Overall, the manuscript exhibits a well-organized structure, introduces novel findings, and aligns its conclusions with existing results and relevant literature.Some minor improvements listed below are expected before the manuscript is formally published 1.The introduction could benefit from a broader context on the impact of viral infectious diseases on humans and animals, as well as the transmission of pathogens from animals to humans.This would provide a comprehensive overview of the significance of the study.2. Figure 8 appears to be of low quality, as if it was copied and pasted.It is recommended that the figure be edited to improve its clarity and presentation.3. The manuscript explores the universal role of p53 in different avian viruses by jointly analyzing RNA-seq and ChIP-seq data.It is noted that the ChIP data is sourced from LMH cells, while the subsequent ALV-J transcriptome data is from DF-1 cells, and the data for IBDV is from bursal cells and IEL-NK cells.The potential impact of these differences on the analysis should be addressed in the Discussion section.4. In Figure 7B, it is advisable to standardize the font to ensure consistency and avoid the use of both bold and non-bold fonts. 5.The manuscript should clearly state the number of experimental replicates performed in each experiment.6.In the Conclusion section, it would be advantageous to underscore the significance of your research findings.Emphasizing how these insights contribute to our understanding of viral infection mechanisms and the development of antiviral strategies would highlight the impact of your work.
Dear Dr. Manjula Kalia, Thank you for your letter dated April 23 rd., 2024!We would like to express our gratitude to you and the reviewers for dedicating your time and effort to reviewing our paper, which is crucial for improving the quality of our manuscript.We have thoroughly considered all comments and suggestions, and accordingly revised the introduction, discussion, and other sections.We also proofread the manuscript to minimize typographical and grammatical errors, and the revised manuscript has been provided as an attachment.
Here below is our description on revision according to the reviewers' comments.
All modifications have been highlighted with yellow background in the revised manuscript.

In the article by Lu Cui and colleagues, the author investigates the molecular networks related to chp53's antiviral activity against IBDV, ALV-J, and ILTV. ChIP-qPCR results indicate that chp53 regulates host cellular metabolism across various viruses.
The authors' answer: Dear Reviewer, Thank you very much for your time and effort spent on reviewing our manuscript!Your insights are invaluable in enhancing the quality of our work.We have addressed each of your comments and suggestions in our revised manuscript, where all modifications have been highlighted for your convenience.Here below is our detailed point-by-point description on revision according to your comments and suggestions.
We appreciate your attention to detail and your contribution to improving our study.

Please incorporate the multiplicity of infection (MOI) of viruses in the materials and methods section.
The authors' answer: Thank you very much for your suggestion!We apologize for the omission of details such as the multiplicity of infection!In the revised manuscript, we have added information regarding the multiplicity of infection and the sample collection time in the Materials and Methods section (page 6, line 113-115).

The reviewer's comment 2:
Figure 7: Please clarify why only metabolic genes were chosen for validation.

The authors' answer:
We sincerely apologize for any confusion caused by our unclear description!The reason we chose to validate only metabolic genes in Figure 7 is because our integrated analysis of ChIP-seq and RNA-seq data revealed that metabolic pathways are commonly regulated by chp53 in chicken cells infected with ILTV, ALV-J, and IBDV.Therefore, seven of these genes were detected to verify whether these conserved metabolic genes are direct targets of chp53.Related explanation has been added in the Result section of the revised manuscript (page 14, line 293-296).We appreciate your attention to this detail and hope this clarification addresses your concern.

The reviewer's comment 3:
For Figure 7, please revise the figure legends to include the significance of the data and the number of experiments conducted.

The authors' answer:
Thank you for your thoughtful reading and valuable suggestions!We sincerely apologize for any confusion that may have arisen from the omission of crucial information in our initial description.We have now clarified the number of biological replicates for each experiment and the significance of the data in the legend of the revised manuscript (page 30, line 643-644).Additionally, information regarding the significance analysis has been annotated in Figure 7.

The reviewer's comment 4:
The authors should elaborate on how the identified pathways are targeted for therapeutic purposes in the context of viral infection.

The authors' answer:
Thank you very much for your suggestions, which have contributed significantly to the improvement of our article!Following your advice, two sections have been added to the Discussion of the revised manuscript, specifically addressing the impact of metabolic pathway alterations on viral replication during viral infection, and the therapeutic use of cellular metabolic pathways in treating viral infections.Following additional references have been added in the revised manuscript (page 18, line 386-400; page 19, lines 402-404).
Thank you once again for your time and effort spent on reviewing our manuscript!Your expertise and insights greatly contribute to the quality of our work.

May 2, 2024 1st Revision -Editorial Decision
Re: Spectrum00309-24R1 (Integrative RNA-seq and ChIP-seq analysis unveils metabolic regulation as a conserved antiviral mechanism of chicken p53) Dear Prof. Hai Li: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.Data Availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact ASM production staff immediately with the expected release date.
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3 .
For Figure 7, please revise the figure legends to include the significance of the data and the number of experiments conducted.4. The authors should elaborate on how the identified pathways are targeted for therapeutic purposes in the context of viral infection.