https://orcid.org/0000-0002-3434-2311
Peer Review History
| Original SubmissionJanuary 18, 2023 |
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Dear Dr. Pessler, Thank you very much for submitting your manuscript "NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner" 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. The reviewers offer several ways in which to strengthen and improve the manuscript conclusions. Please address all of the reviewer comments in a revised manuscript. Experiments with an influenza virus minigenome system are encouraged but would not be required for resubmission. 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. When you are ready to resubmit, please upload the following: [1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. [2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. 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, Jacob S. Yount Academic Editor PLOS Pathogens Meike Dittmann Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************** The reviewers offer several ways in which to strengthen and improve the manuscript conclusions. Please address all of the reviewer comments in a revised manuscript. Experiments with an influenza virus minigenome system are encouraged but would not be required for resubmission. 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 life cycle of influenza A virus (IAV) involves intricate interplay with host cellular factors. Due to the drug-resistance issue of current available anti-IAV drugs that all target viral proteins, it will be highly desired to develop host-directed antiviral drugs. In addition to the well-characterized antioxidative and anti-inflammatory effects of NRF2 activators, they also broadly interfere with the replication of RNA and DNA virus, including IAV. However, the exact mechanism of the antiviral activity of NRF2 activators is largely unclear. The authors in this study endeavored to clarify the underlying anti-IAV mechanisms of four compounds (4OI, BARD, SFN, SEL). They found that although acting as NRF2 activators, the antiviral effect of these compounds on IAV replication was NRF2-independent. Instead, their anti-IAV activity was correlated with their ability to inhibit the nuclear export of vRNP complex. Structural modeling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 critical amino acid Cys528. They concluded that the NRF2 activators inhibit IAV replication by interfering with the nuclear export of viral RNPs in an XPO1-dependent manner. However, some comments have to be addressed to improve the manuscript. Reviewer #2: In this manuscript, Waqas et al. seek to shed light on the mechanisms regulating the antiviral effects of three NRF2 activators on IAV. They show that 4OI, SFN and BARD, as well as the potent CRM1 inhibitor SEL, inhibit more or less efficiently the nuclear export of IAV vRNPs. Surprisingly, they show that the compounds target, interact and probably inactivate CRM1, and that this inhibition is NFR2 independent. More interestingly, they provide ligand-target modeling data suggesting that the three NRF2 activators interact with different energies with the XPO1 NES-binding hydrophobic pocket. Moreover, the XPO1 inhibitor SEL can potentially interact in the same way with the active site of KEAP1, which is the main target of 4OI, SFN and BARD. ********** 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. The authors examined three NRF2 activators, and found that the anti-IAV activity was correlated with their binding energies to XPO1. Are there any other NRF2 activators, if so, whether their anti-IAV activity all depends on their ability to bind XPO1 rather than acting as NRF2 activators. Is the antiviral effect of these NRF2 activators specific to IAV? How about their effect on other viruses that do not have a phase of replication in the nucleus? 2. The authors demonstrated the inhibitory effect of these NRF2 activators on the nuclear export of vRNP complex of IAV. How about their effect on the nuclear export of host cellular proteins. To exclude potential toxic effect, the safe concentration range of the compounds in cells should be provided. 3. To further confirm the conclusion that the four compounds inhibit nuclear export of vRNP complex, the authors had better exclude the possibility that these compounds may affect the vRNP complex activity of IAV by using a minigenome assay. Reviewer #2: 1) Experiments and Figure 3J: - As NRF2 activators likely display their effects by binding and inactivating KEAP1, the expression of the mRNA of the latter should also be checked and provided in the heat map for the different conditions. 2) Experiments and Figure 4: - The authors show that the NRF2 KO cells support significantly higher viral titers, which are still more or less reduced in the presence of the four compounds. They should again analyse the mRNA amounts of KEAP1 and if they remain unchanged, the authors should knock-down KEAP1 with specific siRNAs and then evaluate the virus titers. According to the results provided in Figure 5 and Figure 6, the authors claim that unlike SEL, BARD is a better ligand for KEAP1 than for XPO1 and if so, in the absence of KEAP1, BARD might become more available for binding and inhibiting XPO1 and would thus reduce more efficiently the viral titers. - Analysing the viral titers in conditions of NRF KO and XPO1 KD will provide additional data on the mechanism of action of the analysed compounds. 3) Experiments and Figure 5A & B: - If 4OI binds, as does SEL, the same active sites on XPO1 and KEAP1, competition experiments between these two compounds should provide additional proofs. ********** 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. The shuttling of vRNP complex between cytoplasm and nucleus is critical for the replication of IAV. To offer informative background information, the authors could present the current research advances in the nuclear import and export of vRNP complex of IAV, especially the host factors involved except for XPO1. 2. Page 5, Lines 143-145: Knock-down of XPO1 did not affect levels of IAV hemagglutinin (HA) mRNA or NP in the absence or presence of the four compounds (Fig. 3D, E). However, in Figure 3E, the intensity of ACTB in lanes 8-10 is lower than those in other lanes. Meanwhile, the expression of NP in SEL-treated XPO1 KD cells is also obviously lower than others. Are the provided images representative? The ratio of NP/ACTB had better to be presented by using the ImageJ software. 3. All compounds reduced intracellular IFN responses. The lower mRNA level of IFIT1 and CXCL10 in the drug treatment group can be associated with the inhibition of IFN pathway by drug itself or the lower virus titer caused by the compound treatment. The ISRE reporter assay had better to be carried out to differentiate these possibilities. 4. Line 360: the authors carried out the immunofluorescence assay at 2, 4, 6 and 8 h p.i. However, the images of NP staining at 2 h p.i. were not shown in Fig. 2C. 5. How was the virus titer determined in Fig. 3F and 4B? What does FFU mean? Reviewer #2: 1) Experiments and Figure 2: - The authors should explain the huge differences in the concentrations of the four compounds used, varying from 0.1 to 100 μM and which, I believe, are related to their cellular toxicity. - In 2C the authors should indicate and discuss the percentage of infected (NP-expressing) cells for each condition, with or without treatment. - IF staining of the different conditions is also needed at 0 hpi, as control. Minor issues: The authors should carefully check the references: references 1 and 14 are identical, the reference Qin et al. in the Supplemental Data is already cited in the main list as n°18… ********** 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: No Figure 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. 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 us at figures@plos.org. Data Requirements: Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5. Reproducibility: To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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| Revision 1 |
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Dear Dr. Pessler, We are pleased to inform you that your manuscript 'NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner' 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, Jacob S. Yount Academic Editor PLOS Pathogens Meike Dittmann Section Editor PLOS Pathogens Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens *********************************************************** Reviewer Comments (if any, and for reference): |
| Formally Accepted |
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Dear Dr. Pessler, We are delighted to inform you that your manuscript, "NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner," has been formally accepted for publication in PLOS Pathogens. We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication. The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly. Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers. Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards, Kasturi Haldar Editor-in-Chief PLOS Pathogens orcid.org/0000-0001-5065-158X Michael Malim Editor-in-Chief PLOS Pathogens |
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