PPATHOGENS-D-25-00934

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion

PLOS Pathogens

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS Pathogens. According to the review comments, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Ke Peng

Academic Editor

PLOS Pathogens

Alexander Gorbalenya

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

Additional Editor Comments :

Dear Dr. Wang,

Thank you very much for submitting your manuscript " SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion" 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 revised version that takes into account the reviewers' comments, particularly comments from Reviewer 3.

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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: The authors have addressed all my comments. I am positively impressed by the quality of the revised manuscript. I do not have any additional concern.

Reviewer #2: Zhang et al. demonstrated in this study that the Golgi apparatus and many other organelles are

disturbed by SARS-CoV-2 infection. They focused on the Golgi apparatus and especially on

TGN46 and GRASP55 which are both affected differently in their level of expression by the

SARS-CoV-2 infection. TGN46 is overexpressed while GRASP55 is decreased in expression.

The authors nicely demonstrated that modulation of both proteins either increased or decreased particles production. They

demonstrated that particle secretion was impacted in absence of TGN46 suggesting that SARS-CoV2 divert specific host pathways for its release at the plasma membrane. GRASP55 depletion at the contrary, increases viral secretion, although it is still unclear how the fragmentation of the Golgi apparatus could play a role in increasing particle secretion. The study is interesting and highlight the involvement of the Golgi apparatus in SARS-CoV2 life cycle with key players identified.

Reviewer #3: SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion

This submission documents Golgi fragmentation in SARS-CoV-2 infected cells, thereby confirming previous reports. The study proceeds by treating infected cells with various chemicals that are known to perturb protein synthesis and trafficking, with focus on BFA, a drug that collapses Golgi and has been tested for inhibition of coronavirus MHV secretion. Unlike MHV, BFA blocked SARS-CoV-2 infection. There are then EM images that are used to support statements that SARS-CoV-2 particles reside in Golgi vesicles. The works then advance to GRASP55 and TGN46 because these two Golgi proteins were discovered as reduced and elevated, respectively, in IFA images of infected cells. GRASP55 was then forcibly increased or decreased by transgene expression or siRNA KD. TGN46 was decreased by siRNA KD. Increased GRASP55 did modestly reduce SARS-CoV-2 infection while decreased GRASP55 increased infection. Decreased TGN46 modestly decreased SARS-CoV-2 infection. From these findings, the authors primary conclusion is that the Golgi is required for SARS-CoV-2 secretion.

The results are interesting and potentially valuable because they address current questions about the morphogenesis and egress of coronaviruses particles from infected cells. This study adds some insights into the possible roles for Golgi vesicles in virus secretion. While the authors have responded to three “review commons” recommendations, there are still several concerns with some data interpretations and conclusions. There are also some suggestions for manuscript modification.

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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 Response)

Reviewer #2: The study was nicely improved from the first version of the manuscript. Most of the experiments suggested, were performed and carefully done. The authors supported their microscopy observations with RT-qPCR, confirmed that their observations were not caused by cell death using LDH assays. The biochemical studies to observe particles secretion and the disturbance caused by the virus in host cell receptor localization at the plasma membrane was convincing.

Reviewer #3: Comments:

1. Much of the report seems to be framed around a prior publication in which secretion of the coronavirus MHV was reported to bypass the conventional Golgi secretory pathway (Cell 183: 1520-1535 e1514). Given this perspective, it would seem that SARS-CoV-2 and MHV should be compared directly. Huh-7 – MHV receptor-positive cells can be infected by MHV.

2. While data do show that GRASP55 increases/decreases will decrease/increase SARS-CoV-2, the effects appear modest. Similarly, decreased TGN46 appears to modestly decrease SARS-CoV-2 infection and in the TGN46 case, this reviewer sees little evidence that the effect is at the level of virus secretion. The data in Figs 5, 6 and 7 should be plotted differently, specifically focusing on viral proteins and infectious virions in media vs. lysates. Plotting media/lysate ratios in the case of TGN46 KD might show that TGN46 is reducing infection at stages that are not related to assembled virion secretion (in particular, Fig 7F and 7OP data are not convincingly showing that TGN46 KD impairs virus secretion). Plotting media/lysate ratios for S and N proteins may more convincingly help to determine whether GRASP55 operates at the level of spike protein incorporation into virus particles. Furthermore, M proteins (not N proteins) are the principal coronavirus proteins that operate in virus assembly and the S and M protein ratios may be more illuminating than S and N ratios.

3. The word “expression” is used throughout the paper in reference to changes in GRASP55 and TGN46 protein levels in virus-infected cells. This gives the impression that virus infections are altering GRASP55 and TGN46 gene expressions to facilitate virus assembly and secretion, but gene expressions were not tested after virus infection. Might the altered GRASP55 and TGN46 levels be due to altered protein stabilities following infection, perhaps due to prior Golgi fragmentations? If this remains a possibility, it should be noted and text changed accordingly.

Minor comments:

1. Figs 1E-H; is GPP130 or GRASP65 being imaged? (legend says GRASP65)

2. Figs 2AB; infection “rates” not measured, rather, percent infected cells are measured – consider rephrasing text and ordinat axes

3. Figs 2AB; should note that many of the chemical conditions lack controls (cannot be certain whether some chemicals are active under any condition). Consider removing the data on those drugs that were found to have no effect on infection and were also not shown to have any effects on events in which they should be effecting changes.

4. Fig 2D; should have a positive control condition for LDH release (this applies to all of the data using LDH release as a measure of compromised cells)

5. Fig 2E; should show intracellular N in comparison to the N in media

6. Fig 2C-G; should include MHV-infected cells as comparative control, especially given the importance of this finding and given that the text highlights potential distinctions between SARS-CoV-2 and the previous findings reporting MHV resistance to BFA.

7. Fig 3A-E; is GRASP65 or GM130 being imaged? (legend says GM130)

8. Fig 3P; can agree that white arrowheads are probably pointing to viral particles, but how can it be claimed that black arrows and arrowheads are pointing to golgi membranes and DMVs, respectively?

9. Fig 4; “down and up-regulate” may not be best terms for results that show host protein levels within infected cells by WB (see major comments)

10. Fig 6; the word “accelerates” in the Fig 6 titles is not appropriate, given that kinetic analyses of virus assembly and secretion were not performed.

11. Fig 7; the word “required” in the Fig 7 title is not appropriate, given the modest effects of TGN46 KD on SARS-CoV-2 infection.

12. Discussion, line 460; “integrity of conventional trafficking pathway is essential for SARS-CoV-2..” This statement is puzzling because the data show that the conventional trafficking pathway is disturbed by Golgi fragmentation.

**********

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: No minor modifications

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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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

PPATHOGENS-D-25-00934R1

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion

PLOS Pathogens

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS Pathogens. 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 Aug 03 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,

Ke Peng

Academic Editor

PLOS Pathogens

Alexander Gorbalenya

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

Additional Editor Comments :

Please replace "Nsps" with "nsps" per standard for coronaviruses (consult doi.org/10.1038/s41564-020-0695-z) 

Journal Requirements:

1) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well. Currently, the order and the recipient of this grant "R35HL171421" is different in both places. Please also ensure that the funders of these grants "R21AI152865" and "R01NS102279" match in both places. 

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 #3: Thank you for responses to my review. While the abundant data, the careful experimentation, and the important topic are all very impressive, I remain skeptical of some data interpretations. Note, for example, the reviewer 3 response #5 that addresses effects of BFA on viral protein levels. The response includes a very clean WB showing that BFA dramatically reduces intracellular N proteins. Therefore it is not surprising that there is less secreted virus in response to BFA. Two points here; first, the WB that is presented in responses to reviewers should be in the paper . Second, perhaps more to the point; the overall findings point to BFA blocking N translation or stability, making it hard to interpret findings in context of virus assembly. The authors claim that BFA is blocking virus "assembly" but the data appear to indicate that the BFA effect is at some earlier infection stage.

The above findings related to BFA and effects on N proteins relates to this reviewer's separate suggestion to plot data in terms of media to lysate (M/L) ratios. If this were done for N proteins, i.e., in Fig 2 (and with the additional WB that was provided in response to reviewers), then the M/L ratios would suggest that BFA is not changing M/L ratios and therefore is not directly affecting virus secretion. Hence I stick with the suggestion that M/L ratios have value in data interpretation. The authors note correctly that both unassembled and virus-assembled proteins are present in lysates, while media contain mostly virus-assembled proteins. However, this is not really a confounder in analysis. If GRASP55 and TGN46 are directly affecting virus assembly and secretion, and not other infection stages, then they would be expected to change viral protein M/L ratios. I think the M/L ratios have value in data interpretation.

**********

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 #3: (No Response)

**********

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 #3: (No Response)

**********

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 #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 Dr.Wang,

We are pleased to inform you that your manuscript 'SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion' has been provisionally accepted for publication in PLOS Pathogens.

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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,

Ke Peng

Academic Editor

PLOS Pathogens

Alexander Gorbalenya

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):