PONE-D-23-35404Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the Epithelial Na+ Channel ENaCPLOS ONE

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"The work was supported by grants No. 101720 and A1-S-8290 from Conacyt Mexico to M-CB and GG, respectively. German R. Magaña Avila is a doctoral student from the “Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM)” and received a fellowship from CONACYT (CVU 942671).

We acknowledge the support of a gift from the Joseph and Bessie Feinberg Foundation, a National Institutes of Health grant (1R21 AI166940-01) and a the Northwestern University Clinical and Translational Sciences Institute (NUCATS) COVID-19 Collaborative Innovation Award to DB."

  

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We acknowledge the support of a gift from the Joseph and Bessie Feinberg Foundation, a National Institutes of Health grant (1R21 AI166940-01) and a the Northwestern University Clinical and Translational Sciences Institute (NUCATS) COVID-19 Collaborative Innovation Award to DB."

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"The work was supported by grants No. 101720 and A1-S-8290 from Conacyt Mexico to M-CB and GG, respectively. German R. Magaña Avila is a doctoral student from the “Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM)” and received a fellowship from CONACYT (CVU 942671).

We acknowledge the support of a gift from the Joseph and Bessie Feinberg Foundation, a National Institutes of Health grant (1R21 AI166940-01) and a the Northwestern University Clinical and Translational Sciences Institute (NUCATS) COVID-19 Collaborative Innovation Award to DB."

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Reviewers' comments:

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Comments to the Author

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

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: In this manuscript by Magaña-Avila et al., the authors investigate whether a competition exists between SARS-CoV2 Spike protein and the alpha subunit of ENaC for cleavage by furin, using a Xenopus laevis coexpression system in vitro (WB and patch clamp) and ENaC-gamma subunit expression in immunofluorescence of lungs from SARS-CoV2-infected K18hACE2 mice. The authors conclude that a bi-directional competition exists for furin cleavage between S protein and ENaC-alpha and that this represents a main mechanism for SARS-CoV2-induced ENaC dysfunction.

Major comments.

1. Data presentation is very poor, in view of a complete lack of detail and of essential controls. As such, Western blots do not include MW markers or loading controls. The immunofluorescence images lack antibody isotype controls and quantification.

2. The conclusion of the authors is not supported by the data provided, rather the opposite is true. An S protein mutant with a deficient furin cleavage site inhibits ENaC-alpha cleavage (MW of band not described!) nearly with the same efficacy as wt S protein (Fig. 1). The described inhibition of S protein cleavage by the presence of ENaC-alpha is not clear and the lane numbers are not indicated.

3. Patch clamp data, which should include an amiloride control, only show a minor decrease in ENaC currents due to co-expression with S protein and only for certain voltages. Here mutant S protein does not affect the currents although in Fig 1 its co-expression clearly inhibits ENaC-alpha cleavage. As such, there is a complete disconnect between data in Figs 1 and 2.

4. The co-expression system is artificial since it ignores the signal transduction initiated by binding of S protein to ACE2 in the lungs. ACE2 downregulation upon S protein binding impairs the ACE/ACE2 balance and can activate PKC, a known inhibitor of ENaC open probability (Bao et al., Am J Physiol Renal Physiol. 2007; Chen et al., Am J Physiol Lung Cell Mol Physiol. 2004).

Reviewer #2: In their study, Magana-Avila et al. report the competitive inhibition of cleavage of the alpha subunit of the epithelial sodiun channel ENaC by coexpression of SARS-CoV 2 spike protein in the Xenopus oocyte expression system. Moreover, the authors find that this coincides with reduced ENaC activity in TEVC experiments. Unfortunately, the authors could not present evidence for the impact in vivo since lungs from infected mice were not available for Western blot.

The study is straightforward and the results are presented in a clear and understandable fashion. I particularly like Fig 4 generating a hypothesis fort he pathophysiological impact of the findings.

I have the following suggestions:

1. Fig 1: why is there inhibition of aENaC cleavage at 0.05 spike protein expression whereas cleavage of spike protein is only detectable at 0.4 spike protein expression?

2. Fig 1: It would be interesting to see data on cleavage of gENaC as well since furin is supposed to cleave gENaC once near the N terminus. I suggest the authors to present data on the cleavage of gENaC using the same antibody as used for IF (Stressmarq SPC-405)?

3. Fig. 1: please indicate molecular size of cleaved aENaC.

4. Fig 2: please give sample traces of amiloride-sensitive currents for all groups at e.g. -140 mV holding potential

5. Fig 3: the background seems to high. Can the authors optimize the conditions by diluting the antibody or improving antigen retrieval? The authors should also give a view with a higher magnification as inset.

Reviewer #3: I have reviewed the manuscript Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the Epithelial Na+ Channel ENaC by Bueno et al.

Authors hypothesize that the S-protein competes for furin clevage with the alpha (and gamma) ENaC subunit in lung and that this could contribute to inactivate ENaC and promote fluid accumulation. The hypothesis is tested in vitro in the Xenopus expression system and in vivo in mice exposed to SARS Cov-2 virus. Oocytes were injected with mRNA for all EnaC subunits and with Sprotein.

Alpha ENaC cleavage and inward amiloride-sensitive currents were attenuated by S-protein overexpression but not in S-protein with mutated furin-cleavage site. Only immunofluorescence was possible in infected mouse lungs. Data indicate that overexpression of S-protein engages furin and leaves alpha ENaC uncleaved.

The idea is good and novel and well presented in the introduction. I have some suggestions that authors could consider in order to improve their manuscript.

1. From which species were the mRNAs that were injected?

2. Why was furin not overexpressed and where does the proposed reaction occur? In the biosynthesis pathway or on the surface? I realize there is a schematic drawing but no data are presented on this issue.

3. Is the amount of furin always the same or in other words, does the alleged competition not also depend on the amount of furin?

4. Can it be excluded that other inherent/endogenous proteases contribe to cleavage?

5. It would strengthen the data to knock out furin in oocytes or at least demonstrate furin.

6. Why was an accepted protease inhibitior not used as a positive control, i.e. aprotinin or alike to test that a similar inhibition as with S-protein overexpression was observed and to map the part of the reaction taking place on the cell surface.

7. How do you compare densitometry across gels on the immunoblots – this should be described better(/in detail.

8. In figure 1, please state the expected migratory pattern of each protein species and how it compares with actual migration and place independent molecular weight markers. A C-terminal antibody against human αENaC would show proteins migrating putatively at 74, 51, and 48 kDa (non-glycosylated). Was that the case ?

9. Authors express alpha, beta and gamma ENaC -but I can only find immunoblotting data on alpha. Why are especially gamma ENaC not evaluated since it also depends on furin for cleavage and an extra cleavage to gain full activity in the intact channel.

10. Frankly, n=3 does not allow meaningful statistical evaluation (fig 1). Statistical methods are not described except for fig 2 data (or I cannot find it). Please state in figure legends.

11. What is “K18hACE2 transgenic mice” -please provide details. How many mice were used in total and how many died before inclusion- these parameters should be reported

12. It is a pity that fresh lung tissue from the mice was not available for immunoblotting, however it would have some value to validate immunofluorescence by running immunoblots on non-infected control mice to demonstrate that the antigen is significantly present in the adult mouse lung. And compared to kidney.

13. In contrast to the immunoblotting where only alpha ENaC data are shown, in immunofluorescence only gammaENaC is shown. Why? Please add data on alpha. In the legend to fig 3 it is stated “ENaC” but not alpha or gamma. Please clarify.

14. The number of the protocol approving the experiments and the date should be given.

15. The magnificantion of the immunofluorescence and/or the resolution in my cope does not allow to see much detail. Could higher magnificantion be shown.

16. Please delete the speculative parts of the discussion on dexamethasone, it is tangential, not tested in the study and although interesting, not relevant in the present context.

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Reviewer #1: Yes: Rudolf Lucas

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-35404R1Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the Epithelial Na+ Channel ENaCPLOS ONE

Dear Dr. BUENO,

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 still raised by the reviewer.

Please submit your revised manuscript by Apr 25 2024 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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• 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.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

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Michael Bader

Academic Editor

PLOS ONE

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Reviewers' comments:

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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)

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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: No

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

Reviewer #1: 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: No

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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

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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: In this revised version, it is appreciated that the authors tried to address the comments made by the three reviewers. However, a lot of issues remain for this reviewer with poorly controlled experiments, doubtful interpretation of results and weak consideration of alternative explanations. Here a summary:

Introduction

1. Not only type 2 but also type 1 AT express ENaC

2. COVID-ARDS is not more inflammatory than non COVID ARDS. As such, listing inflammation as the main cause of edema formation is questionable. Alveolar-capillary barrier dysfunction is also very important and is not discussed.

Results

1. As brought up before, the mutant S protein has only a two-fold reduced capacity to inhibit ENaC-alpha cleavage as compared to the WT S protein, strongly questioning the main hypothesis.

2. As brought up before, in Fig 2, it is not acceptable to just subtract the amiloride data from the rest. All voltage current tracers should be shown.

3. The fact that the mutant S protein does not significantly differ from the WT S protein in the patch clamp study, together with a lack of effect of SARS-CoV2 infection on ENaC-gamma expression in lungs in Fig. 3 highly questions the relevance of the proposed hypothesis.

4. Lack of an essential isotype control Ab in Fig. 3. Referring to one manuscript where the anti-ENaC-gamma antibody was used does not suffice.

5. The manuscript now mentions furin-like proteases throughout, but uses an S protein mutant that has a specific deletion in the furin cleavage site. This is confusing. As suggested by another reviewer, why didn't the authors overexpress furin or used furin inhibitors in their studies? This weakens the aim of the study.

6. Whereas SP-C is a specific marker for type 2 alveolar epithelial cells, SP-A, used in Fig 3, is not, since it can also be secreted by non-ciliated bronchiolar cells, submucosal gland and epithelial cells of other respiratory tissues, such as the trachea and bronchi (Carreto-Binaghi LE, Aliouat el M, Taylor ML. Surfactant proteins, SP-A and SP-D, in respiratory fungal infections: their role in the inflammatory response. Respir Res. 2016 Jun 1;17(1):66. doi: 10.1186/s12931-016-0385-9. PMID: 27250970; PMCID: PMC4888672).

Discussion

1. PKC activation is being discarded by the authors as an explanation for the ENaC- inhibitory effect of the S protein. However, a recent paper performed in primary human cells showed that the receptor binding domain of S1 -so the form after cleavage by furin- is highly efficient in inhibiting ENaC activity, albeit in a different cell type (human lung MVEC). The oocyte expression system used by the authors or by the Clark paper they refer to does not allow to investigate this, since there is no human ACE2 expression. If the cleaved S protein binds to hACE2, this increases ACE and subsequent PKC activity. As such, if the machinery to mediate S protein-induced PKC activation is missing no valid conclusions can be made.

In conclusion, the authors did not convincingly demonstrate that SARS-CoV2 S protein inhibits ENaC-alpha and gamma cleavage mainly by hijacking furin. There data rather indicate that this pathway is not important.

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Reviewer #1: No

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Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the Epithelial Na+ Channel ENaC

PONE-D-23-35404R2

Dear Dr. BUENO,

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.

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