TRPV4-mediated calcium influx governs temperature-dependent SARS-CoV-2 replication

PLOS Pathogens

Dear Dr. Ichinohe,

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Kind regards,

Yaw Shin Ooi, PhD

Academic Editor

PLOS Pathogens

Ashley St. John

Section Editor

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments (if provided):

Thank you for submitting the revised version of your manuscript. The revised manuscript has now been evaluated by the reviewers.

The reviewers acknowledge that the revision has addressed many of the concerns raised previously. In particular, the evidence supporting a role for intracellular calcium in SARS-CoV-2 replication and TRPV4 as a contributor to this process is considered convincing. However, concerns remain regarding the central mechanistic conclusion that the TRPV4–Ca²⁺ axis underlies temperature-dependent SARS-CoV-2 replication.

In preparing your revision, please carefully consider whether the overall framing and conclusions of the manuscript are fully supported by the experimental evidence. If the current data do not directly establish the mechanistic link between temperature and the TRPV4–Ca²⁺ axis, the title, abstract, Results, and Discussion should be revised accordingly to ensure that the conclusions accurately reflect the scope of the data. Please also address all reviewer comments in a detailed point-by-point response.

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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: I sincerely thank the authors for thoroughly addressing all of my concerns.

Reviewer #2: Kobayashi and Abe et al. identify TRPV4, a temperature-dependent calcium channel, as a major contributor to the temperature-dependent replication of SARS-CoV-2 at physiologically relevant temperatures: 34°C, representing the upper respiratory tract; 38°C, representing core body temperature; and 39°C, representing a fever-like condition. The authors show consistent trends across three viral variants. Their mechanistic evidence is supported by a comprehensive set of experiments focused on calcium influx. Specifically, they use a TRPV4 agonist at 34°C to increase viral replication and a TRPV4 antagonist at 37°C to inhibit replication. To further support calcium influx as a key factor in temperature-sensitive SARS-CoV-2 replication, they use the broad calcium ionophore ionomycin and the calcium chelator BAPTA-AM to manipulate intracellular calcium levels, producing results consistent with the TRPV4 experiments. The controls are thorough and well designed, including assessments of drug toxicity, potential off-target effects on proteins known to affect SARS-CoV-2 infection such as ACE2 and TMPRSS2, and treatment effects at the relevant temperatures. In addition, the authors perform genetic siRNA knockdown experiments and show that SARS-CoV-2 replication is reduced following TRPV4 knockdown at 37°C. The authors also perform in vivo experiments in a hamster model. Treatment with the general calcium channel blockers cyclosporin A and manidipine has a significant effect on survival. In contrast, a TRPV4-specific inhibitor shows only a modest protective effect in animals. Overall, the data are well controlled and carefully executed, with substantial experimental replication.

Reviewer #3: This manuscript identify the TRPV4 play an important role during the SARS-CoV-2 infection cycle by regulation calcium concentration as the thermosensitive channel. Meanwhile, the authors tested the therapeutic potential of targeting calcium channel in hamster. However, the mechanistic interaction between the TRPV4 and SARS-CoV-2 need further clarification and validation. There have been one review about the host cell calcium dynamics and SARS-CoV-2 infection (https://doi.org/10.1016/j.ceca.2022.102637). Several issues should be further explored or clarification.

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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 additional experiments required from my side.

Reviewer #2: n/a

Reviewer #3: The authors should be cautious with the quantification results of viral N gRNA, usually the RNA copies are hundreds or thousands times higher than the viral titers, but it is opposite in this study.

The authors investigated SARS-CoV-2 replication under different temperature (34 °C ,37°C , and 39 °C) ; however, the impact of temperature on cellular state was neither measured nor discussed, as most mammalian cell lines exhibit growth limitations at 39°C. Notably, it is worth noting that both TRPV4 agonists and inhibitors had minimal effects on SARS-CoV-2 replication.

It is interesting to sequence the genome of the cold-adapted SARS-CoV-2, then check the sequence mutations of the cold-adapted variants and confirm the locations of the mutations. This may help us identify which proteins play important functions during the temperature-dependent process. The authors should discuss this point in the manuscript.

In the Figure 2E and 3F, the virus titer value of PFU for the control group looks same, are they independent experiments or use the same control?

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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: The calcineurin-related data are less clear. Although these experiments appear to be well executed, they do not currently add substantial mechanistic insight. If the authors intend to link calcium influx to downstream calcineurin signaling as an in-depth mechanism, additional experiments will be needed to support this connection more directly. In its current form, this section introduces some confusion rather than strengthening the overall manuscript.

Although the evidence that calcium influx affects SARS-CoV-2 replication is convincing, and the role of TRPV4 appears significant, the authors should be cautious in claiming that temperature-dependent SARS-CoV-2 replication is driven specifically by TRPV4. The data support TRPV4 as an important contributor, but none of the experiments demonstrate complete rescue to the level observed at 37°C. Furthermore, the in vivo experiment of TRPV4 inhibitor is not convincing. The survival curves have very minor differences between the mock and treatment groups. Why are there only days 3 and 5 weight loss shown? Why was only day 3 titer significant?

Overall, the manuscript is well done and just requires some changes in wording to avoid over claiming the significant. For instance: in the title, “governs” may be too strong, “influence” or similar terms may be more appropriate.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: Yes:  Long Ping Victor Tse

Reviewer #3: No

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Figure resubmission:

Reproducibility:

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Dear Dr. Ichinohe,

We are pleased to inform you that your manuscript 'TRPV4-mediated calcium influx contributes temperature-dependent SARS-CoV-2 replication' 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.

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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,

Yaw Shin Ooi, PhD

Academic Editor

PLOS Pathogens

Ashley St. John

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

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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 #2: I have no additional comment.

Reviewer #3: The authors have answered the questions properly.

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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 #2: (No Response)

Reviewer #3: The authors have answered the questions properly.

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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 #2: (No Response)

Reviewer #3: No

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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 #2: Yes:  Long Ping Victor Tse

Reviewer #3: No