PPATHOGENS-D-25-00688

Identification of picornavirus proteins that inhibit de novo nucleotide synthesis during infection

PLOS Pathogens

Dear Dr. van Kuppeveld,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 30 days Jun 23 2025 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Peter Sarnow

Academic Editor

PLOS Pathogens

Sonja Best

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

Journal Requirements:

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: Lonneke Nouwen, Esther A Zaal, Inge Buitendijk, Marleen Zwaagstra, Chiara Aloise, Arno L.W. van Vliet, Jelle G Schipper, Alain Van Mil, Celia R Berkers, and Frank J.M. van Kuppeveld. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

2) We ask that a manuscript source file is provided at Revision. Please upload your manuscript file as a .doc, .docx, .rtf or .tex. If you are providing a .tex file, please upload it under the item type u2018LaTeX Source Fileu2019 and leave your .pdf version as the item type u2018Manuscriptu2019.

3) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- TM on page: 24.

4) Your manuscript's sections are not in the correct order.  Please amend to the following order: Abstract, Introduction, Results, Discussion, and Methods

5) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines: 

https://journals.plos.org/plospathogens/s/figures

6) We have noticed that you have uploaded Supporting Information files, but you have not included a list of legends. Please add a full list of legends for your Supporting Information files after the references list.

7) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager. 

Potential Copyright Issues:

i) Figure 4B. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art:

- https://commons.wikimedia.org

- https://openclipart.org/.

8) We note that your Data Availability Statement is currently as follows: "All relevant data are within the manuscript and its Supporting Information files.". Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data: 

1) The values behind the means, standard deviations and other measures reported;

2) The values used to build graphs;

3) The points extracted from images for analysis..

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. 

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

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: Nouwen and colleagues present a comprehensive investigation of metabolic changes in cells infected with picornaviruses focusing on the nucleotide and amino acid synthesis pathways. Picornaviruses are known to induce rapid and profound changes in cellular metabolism, but the mechanism and significance of many of such infection-induced perturbations remain poorly understood. Using metabolic tracing with different labeled compounds which allowed complementary assessment of diverse metabolic pathways, the authors show that infection with Coxsackievirus B3, an enterovirus, and encephalomyocarditis virus, a cardiovirus, inhibits de novo nucleotide synthesis but activates the nucleotide salvage pathways. Using elegant replication systems where they could inactivate and substitute specific viral proteins they convincingly demonstrate that enterovirus protease 2A and cardiovirus leader protein L are largely responsible for the observed changes in nucleotide metabolism. They further speculate that given that both proteins induce inhibition of nucleocytoplasmic trafficking, albeit by different mechanisms, the latter may be required for the inhibition of de novo nucleotide synthesis. The major findings obtained in HeLa cells were also corroborated in a more physiological system of cardiomyocytes differentiated from pluripotent cells.

The experiments are performed on an excellent technical level and will represent an important resource for everyone interested in metabolic changes in infected cells.

Reviewer #2: Building upon their previous report (citation #34; PLos Path 2024), Nouwen and colleagues report that the 2A protease of CVB3 and the leader protein of EMCV inhibit de novo nucleotide synthesis. These conclusions are substantiated by experiments exploiting state-of-the-art isotope tracing metabolomics and a clever panel of genetically engineered coxsackieviruses (Fig. 4B): CVB3, CVB3 with a debilitating 2A protease mutation, and derivatives thereof expressing the EMCV leader protein or the FMDV leader protease. As the authors highlight (Fig. 4A), the EMCV and FMDV leader proteins share some phenotypic traits with CVB 2A protease, allowing the authors to test whether previously established phenotypes of CVB3 2A protease (nucleocytoplasmic trafficking disorder, cap-dependent host translation shut-off via eIF4G cleavage or IFN suppression) influence de novo nucleotide synthesis. Intriguingly, CVB3 2A protease and EMCV leader protein inhibit de novo nucleotide synthesis, while FMDV leader protease does not. The inhibition of de novo nucleotide synthesis was clear in CVB3-infected HeLa R19 cells (where de novo nucleotide synthesis is a normal aspect of cellular metabolism). However, similar phenotypes were not completely evident in human induced pluripotent stem cell-derived cardiomyocytes (where a lower rate of de novo nucleotide synthesis confounds some comparisons with phenotypes in HeLa cells).

The authors conclude that the CVB3 2A protease and the EMCV leader protein, but not the FMDV leader protease, inhibit de novo nucleotide synthesis. Furthermore, they conclude that the nucleotide salvage pathway is activated during CVB3 infection, providing a source of nucleotides for virus replication. The authors cautiously suggest that nuclear pores and/or nucleocytoplasmic trafficking could be linked to de novo nucleotide synthesis, and modifications of nuclear pores by CVB3 2A protease and the EMCV leader protein could disrupt and/or relocalize nuclear pore adjacent enzymes involved in de novo nucleotide synthesis.

Critique: This is a novel investigation with important insights into nucleotide metabolism during enterovirus infections. It appears that the nucleotide salvage pathway, rather than de novo nucleotide synthesis, is activated during CVB3 infection, providing a source of nucleotides for virus replication. The authors provide strong evidence to show that the CVB3 2A protease and the EMCV leader protein, but not the FMDV leader protease, inhibit de novo nucleotide synthesis. However, it is unclear whether the inhibition of de novo nucleotide synthesis by viral proteins benefits virus replication, or is otherwise biologically relevant in the context of enterovirus infections in vitro or in vivo. Is the inhibition of de novo nucleotide synthesis biologically relevant, or an incidental phenotype?

Reviewer #3: This manuscript focuses on the nucleotide metabolism control during picornavirus infection. Specifically, the authors used isotope labeling techniques in metabolomics to intricately trace the pathways of nucleotide metabolism to identify how a specific viral protein 2A in Cocksackie B3 virus, controlled these pathways. They discovered that the wild type virus down regulated de novo biosynthesis pathways and this down regulation was dependent upon 2A activity. Additionally, they show that this phenotype is reproducible in additionaly cell types and across picornaviruses.

Isotope tracing is extremely challenging to do, and the authors have carried out well-controlled, systematic analyses that are very in-depth and elegantly designed. The results are very clear and informative for the field and I see no problems. Issues below are to improve clarity.

minor issues:

In the discussion, it would be good to hear hypotheses about why these viruses might prefer to down regulate de novo synthesis and instead rely on salvage pathways of nucleotide metabolism.

CAD is not defined.

line 508, sentence is not complete.

**********

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: One missing point that should be addressed, at least in the Discussion, is the significance of the metabolic changes observed for the viral infection. Given that the viruses defective in the inhibition of de novo nucleotide synthesis can replicate reasonably well, and that the magnitude of the effect is different in different cell types, do the authors consider the variations in nucleotide metabolism to be essential for the development of infection, especially in the in vivo conditions? Can the inhibition of the nucleotide salvage pathway on the development of infection be tested?

Reviewer #2: 1. Biological significance. Is the inhibition of de novo nucleotide synthesis advantageous for enteroviruses, or an incidental phenotype? It is unclear whether the inhibition of de novo nucleotide synthesis by viral proteins benefits virus replication, or is otherwise biologically relevant in the context of enterovirus infections in vitro or in vivo.

Reviewer #3: None

**********

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: Line 188 of, not off

Line 233. The expression “favorable function upon expression” is vague and unnecessary. It is sufficient to state that mutations in 2B result in the replication delay.

Reviewer #2: 1. Line 151 and elsewhere throughout the manuscript. Clarify the MOI used for experimental infections. Virus titers were obtained by limiting dilution (TCID50 per ml); Does 5 TCID50 per ml = 5 TCID50 per cell for experimental infections?

2. Line 177. ...reduced or deduced?

3. Theoretical consideration. Are there any limiting metabolites evident at any point during infection? Are cells metabolically exhausted at any point during infection, where low amounts of one or another metabolite could limit viral gene expression and replication?

4. Mechanistic Gaps. It remains unclear how CVB3 2A protease and the EMCV leader protein inhibit de novo nucleotide synthesis. The discussion provided some very nice speculation (for future studies) to address mechanistic gaps.

Reviewer #3: minor issues:

In the discussion, it would be good to hear hypotheses about why these viruses might prefer to down regulate de novo synthesis and instead rely on salvage pathways of nucleotide metabolism.

CAD is not defined.

line 508, sentence is not complete.

**********

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

Reviewer #3: 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.]

Figure resubmission:

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. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Prof. Dr. van Kuppeveld,

We are pleased to inform you that your manuscript 'Identification of picornavirus proteins that inhibit de novo nucleotide synthesis during infection' 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,

Peter Sarnow

Academic Editor

PLOS Pathogens

Sonja Best

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):