Peer Review History
| Original SubmissionAugust 9, 2021 |
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PONE-D-21-25746 Elevated anti-interferon activity of lyssavirus phosphoproteins in bat compared to human cells PLOS ONE Dear Dr. Müller, 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 by two experts during the review process. These points include, but are not limited to, clarification and quantification of P protein Western blotting results and clearer justification of the authors' main conclusion (higher anti-interferon activity of P proteins in bat relative to human cells). Please submit your revised manuscript by Oct 15 2021 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. Please include the following items when submitting your revised manuscript:
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PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 3. 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 ********** 4. 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 ********** 5. 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: PONE-D-21-25746, Papies et al.: Elevated anti-interferon activity of lyssavirus phosphoproteins in bat compared to human cells Bats were reported to carry highly pathogenic zoonotic viruses often without showing symptoms or suffering from disease. This is largely attributed to an enhanced (IFN) antiviral and decreased inflammatory response in bats. In the present work, the authors address the question whether the phosphoprotein (P) of Lagos bat lyssavirus (LBV), a rabies-related virus, has an enhanced anti-IFN activity in cells of their natural bat host species as compared to human cells. This might indicate adaptation to the enhanced antiviral response in bats. To this end, they generated Eidolon bat cell lines and novel bat IFN-ß reporter plasmids and provide their characterization, including IFN-ß response and permissivity of cells for a LBV isolate (Fig. 1, S1), and response of the novel plasmids in bat cells to RNA- and viral PAMPs (Fig. 3). In reporter gene experiments – and somewhat contrary to the title - they observe roughly comparable inhibition of human and bat IFN-ß promoter activation by control rabies virus P proteins, while the inhibitory activity of LBV P protein in human cells is markedly reduced and, if at all, only marginally better in bat cells (significant in 1 of 2 cell lines)(Fig. 4). While most of the provided experiments were performed and presented adequately, I do not agree with the conclusions emphasized in the title and discussion of “enhanced anti-interferon activity of lyssavirus phosphoproteins in bat compared to human cells”. In my opinion the data indicate that the activity of LBV P (and only with respect to IFN-ß promoter activation) is not enhanced in bat cells, but rather reduced in human cells. Note that RABV P and DUVV, which is another bat lyssavirus, perform well in both human and bat cells, the exception is LBV P showing defects in human cells. Major: 1. Apart from the “enhanced in … bat” issue, the title is too general and broad, it should be limited to: Elevated IFN-ß inhibitory (not: anti-interferon) activity of activity of Lagos Bat lyssavirus phosphoproteins in bat compared to human cells. Note that DUVV is also a bat lyssavirus, and RV a lyssavirus, and the present title would misleadingly implicate a common general feature of lyssaviruses. In addition, only IFN-ß promoter activation was studies in bat cells, not anti-IFN activity. 2. With respect to a general “anti-interferon” activity as implied in title, Fig. 2 is the most meaningful. There it is shown in human cells that LBV P seems to have deficits in IFN-ß promoter inhibition (Fig. 2A), while it catches up or even overtakes RVP when it comes to ISRE inhibition (Fig. 2B), i.e., regarding IFN induction and IFN-STAT signaling is regarded in total. Enhanced STAT inhibition would be very exciting and could be determined in a simple and specific ISRE experiment, just by adding exogenous IFN rather than transfect RNA. In bat cells, if bat ISRE reporters are not available, RT-PCR for ISGs could be done. Such simple experiment would clarify whether LBV P is better adapted to antagonize bat anti-interferon responses, or not. 3. S2B Fig.: There seems to be something wrong with the P proteins in WB. First, RVP is of different size in HEK-293T (<<40 kDa) and other cell lines (>=40 kDa): check marker. More worrying is the identity of the proteins, as in HEK-293T and EidNi/41.3 LBV-GH-P is smaller than the other lyssavirus P proteins, while in A549 and EidLu/20.2 the small protein is LBV-Nig P. On first sight, it appears that LBV-GH and LBV-Nig proteins were interchanged. Please check, and make sure correct designation in the other experiments and tables. Although this does not severely affect the overall conclusion, it is not suitable to inspire confidence in the experimental diligence. 4. S1 Fig. IFN competence and antiviral activity of IFN in human A549 (A) and EidLu/20.2 bat cells (B). Unexpectedly, and in contrast to the popular view, IFN bioactivity is way lower in bat cells. Irrespective of this, however, the somewhat (2-fold) lower replication of LBV in A594 (Fig. 1) is not attributed to the way higher bioactivity of A549, but rather to an allegedly higher LBV anti-IFN activity in EidLu/20.2. Please reconsider such reasoning. In addition, in Fig. 1 please include LBV infectious titers, which are more relevant for appreciation of the full extent of antiviral activity than genome copies. Minor: l. 383ff: move promoter sequence IDs to Materials and Methods, in addition, provide sequence IDs of P proteins used. l.47: replace frequently with sporadically (human transmissions) l. 388: Yinpterochiroptera is mentioned in the text, please indicate suborder in Fig. 3 l. 412: replace Fig 3C with Fig 3D Reviewer #2: The authors outline their new luciferase induction model for analysis of IFN-beta kinetics in bat cells. The details are clearly worked out and explained with some key points that are relevant to the field. The claims based on differences in IFN induction between different RVP/LBV constructs are based on western blot images the authors claim are equally expressed. P is clearly differentially expressed between constructs and cell lines and these needs to be quantified and normalized to housekeeping before any claim on differences can be made. It looks like there are large differences in LVP vs RVP expression that would affect these results. For figure 4 internally there are statistical differences between constructs within the cell line but in the results the authors also compare results across cell lines. To prove this is relevant/significant the data needs to be directly compared with statistics between bat vs human for example. It is good they have two bat and two human cell lines but the differences are still quite small and ideally a third bat and human cell line should be included. Failing that, the authors need to clearly state that with such a limited number of cell lines all affects could simply be cell-line specific and not related to species differences, highlighting the limitations in the study. While the authors carefully worked out promoter/concentration differences in the EiD cell lines the authors need to mention in the discussion how this is all done with artificial stimulation (transfected rna / rvfv reporter etc) and how it may differ with real infection. They should also indicate references etc showing how LBV/RBV infection would be expected to activate the same RIG-I/MDA-5 signaling pathways as VSV RNA. There is also limited discussion on the differences between IFNs and if other (for example alpha, omega, kappa) promoters are expected to be affected in a similar fashion. There was limited discussion on ISG54 promoter whereby no species differences were observed and how what may be more relevant during infection - P's effect on IFN-b promoter compared to direct on ISG induction visa ISRE elements. (a comparison of ISG54 induction in the bat cells would be ideal). With that in mind there are other studies showing high ISG induction and characterizing IFN induction (the authors own work, ref. 56 and also https://pubmed.ncbi.nlm.nih.gov/33147460/) that are highly relevant to this study and should be discussed. Additionally there is data showing unique bat-specific IRf3 phospho sites that may be relevant to activation (and viral-induced inhibition from TBK1) that could be discussed - https://www.cell.com/iscience/fulltext/S2589-0042(20)30142-5. ********** 6. 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 [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. 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| Revision 1 |
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Reduced IFN-ß inhibitory activity of Lagos bat virus phosphoproteins in human compared to Eidolon helvum bat cells PONE-D-21-25746R1 Dear Dr. Müller, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Zheng Xing 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 #1: All comments have been addressed ********** 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: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ********** 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 ********** 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 ********** 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: (No Response) ********** 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 #1: Yes: Karl-Klaus Conzelmann |
| Formally Accepted |
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PONE-D-21-25746R1 Reduced IFN-ß inhibitory activity of Lagos bat virus phosphoproteins in human compared to Eidolon helvum bat cells Dear Dr. Müller: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Zheng Xing Academic Editor PLOS ONE |
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