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Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.

PLOS Pathogens

Dear Dr. Singh,

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Kind regards,

Yong-Hui Zheng, Ph.D.

Guest Editor

PLOS Pathogens

Michael Letko

Section Editor

PLOS Pathogens

 -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

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Michael Malim

Editor-in-Chief

PLOS Pathogens

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Additional Editor Comments :

Please revise your manuscript based on the two reviewers' comments. Please keep in mind that additional experiments must be conducted to address their concerns.

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Ragini Agrawal, Virender Pal, Suhas KS, Gopika Menon, Inder Raj Singh, Nitish Malhotra, Naren CS, Kailash Ganesh, Raju Rajmani, Aswin Seshasayee, Nagasuma Chandra, Manjunath Joshi, and Amit Singh. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

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: Ragini Agrawal et al. demonstrate that H2S gas participates in the modulation of SARS-CoV-2 infection by suppressing virus replication. The down-regulation of H2S-producing enzymes, including CBS, CTH, and 3-MST, has been observed to be associated with virus replication. The use of GYY4137, a slow-releasing H2S donor, for chemical supplementation of H2S was found to diminish virus replication. This was achieved by inducing the Nrf2/Keap1 pathway, restoring redox balance and carbon metabolites, and potentiating mitochondrial oxidative phosphorylation.

This study is well-written and nicely organized and presented, with carefully crafted figures. However, I believe there are a number of minor to moderate changes that could be introduced to improve the manuscript.

Reviewer #2: In this manuscript, the authors investigate the role of H2S on SARS-CoV-2 replication. Previous research from this group and others has demonstrated the antiviral properties of H2S on respiratory pathogens. The authors conclude that SARS-CoV-2 limits intracellular H2S in infected cells and H2S inhibits viral replication through modulation of the metabolic pathways. Further increasing H2S through pharmalogical intervention blunted viral replication in the lung of infected mice and hamsters, leading to reduced pathology and respiratory distress.

This is a very well written manuscript with several different lines of evidence that support the authors conclusion. I have noted below one alternative hypothesis that should be addressed.

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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: 1.Previous papers (PMID: 32515982) have shown that H2S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2, inhibit SARS-CoV-2 replication by attenuating virus assembly/release, and protect SARS-CoV-2-induced lung damage by suppressing immune response and inflammation development. I suggest the authors to detect the expression of ACE2 and TMPRSS2 in the paper to exclude its function in the decreased virus replication.

2.In Fig 5I: “We noticed that treatment with GYY4137 specifically reversed the effect of SARS-CoV-2-HK infection on the expression of genes associated with the oxidoreductase complex and Nrf2-antioxidant pathway (Fig. 3I)”. Were the oxidoreductase complex also analyzed in the transcriptional data?

3.The full dataset of the RNA-seq needs to be provided in the supplemental information.

4.In line 247-250: “Consistent with this finding, we observed down-regulation of antioxidant genes (e.g., glutathione peroxidases [gpx4], peroxiredoxins [prdx6], hemoxygenase (hmox1], catalase [cat], and thioredoxins [txnrd1])) regulated by nuclear factor erythroid 2-related factor 2 (Nrf2)-a central controller of cellular resistance to redox stress[34]”. This concluded from the experiment or Reference 34? In addition, I could find prdx6 in the results throughout the manuscript.

5.In line 255-257: “As with our RT-qPCR data, infection with SARS-CoV-2 reduces the expression of genes associated with sulfur metabolism, which include H2S biogenesis (Fig. 3D).” The results presented in Figure 3D are inconsistent with the description.

6.The authors should clarify why the Nrf2 gene was not included in the RNA-seq data, despite its expression being detected by WB.

7.According to the results, H2S suppression of SARS-CoV-2 proliferation is associated with the expression of the Nrf2/Keap-1 regulon, redox metabolites, and mitochondrial function. Therefore, what is the relationship among these factors? Which one serves as the primary influencing factor?

8.The authors should clarify the rationale behind using different treatments in Figure 3 and Figure 4. Additionally, they should discuss whether this discrepancy in treatment affects the results obtained.

Reviewer #2: The authors conclude that H2S suppresses viral replication through modulation of host cell metabolic pathways. However an alternative conclusion from the data could be that H2S inhibits viral entry and cells are less infected. All of the experiments in this manuscript pretreat the cells/mice with higher levels of H2S, either through treatment with a slow H2S release chemical or genetic manipulation of the H2S synthesis pathways. To address this alternative hypothesis, I suggest the following experiment. Infect cells at a high MOI (3-5). Wash cells after one hour and treat with H2S. Monitor viral growth kinetics.

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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: 1.Fig. 2A: The label for Fig. 2A is missing in the Results section.

2.In line 225-232: I was unable to locate the genes in Fig 3, and the reference was not provided either. Kindly provide a detailed description of it.

3.Fig. 6I: Based on the text, there should be a label (7) missing in Fig. 6I. Furthermore, please clarify the meaning of 'BV' in Fig. 6I?

4.In lines 1314-1315: “(B) denotes bronchial lumen and (AS) denotes alveolar space”. These annotations may require revision.

Reviewer #2: - Fig 1H. Violin plots are distracting. Please show scatter plot of data points.

- Fig 6B right panel. Y-axis title should read n gene copy number. Also how is this data normalized?

- Fig 6 general: Make groups across different panels the same color. For example SARS-CoV-2 + GYY4137 is red in 6A-E, then green in 6F-H.

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Dear Dr Singh,

We are pleased to inform you that your manuscript 'Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Yong-Hui Zheng, Ph.D.

Guest Editor

PLOS Pathogens

Michael Letko

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 adequately addressed the concerns and suggestions raised in the initial review. The revisions have improved the manuscript's clarity, rigor, and overall quality. The study provides valuable insights into the role of H₂S in modulating SARS-CoV-2 infection, particularly in suppressing viral replication through the Nrf2/Keap1 pathway, redox balance restoration, and mitochondrial oxidative phosphorylation. This study makes a meaningful contribution to understanding host-directed antiviral strategies and highlights the therapeutic potential of H₂S donors in SARS-CoV-2 infection.

The manuscript remains well-organized, with logical flow and carefully designed figures. Since the authors have satisfactorily addressed all previous concerns, I recommend accepting it in its current form. No further revisions are required.

This review acknowledges the improvements made and confirms that the manuscript is now suitable for publication.

Reviewer #2: Thank you for addressing all critiques.

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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)

Reviewer #2: N/A

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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)

Reviewer #2: N/A