Dear Prof Liao,

Thank you very much for submitting your manuscript "Inhibition of Anti-viral Stress Granule Formation by infectious bronchitis virus endoribonuclease nsp15 Ensures Efficient Virus Replication" 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 significantly-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

I am returning the manuscript with comments from four reviewers who provide differing views of the manuscript. Overall the reviewers viewed the study as an interesting addition to current knowledge about CoV regulation of stress granules, identifying a role for nsp15. In addition, linking IBV regulation of stress granules with interferon signalling supports recent developments connecting these important signalling pathways and reports of antiviral stress granules induced in response to other viruses. However, the reviewers raised some valid questions about contradictions in the mechanism of action of nsp15 that are not fully addressed in the manuscript currently and an important missing control experiment. They also highlighted that more complete discussion of the data presented in the context of existing understanding about the role of nsp15 is needed. Based on this, I am recommending Major Revisions. I am sorry I cannot be more positive at the moment; however, we are looking forward to receiving your revision.

Please read through and address the concerns of the reviewers and in particular the following points.

Major changes:

1. The reviewers’ major concerns will be addressed by investigating further the mechanism of nsp15 action, as outlined in the three options in the discussion. Particularly what nsp15 is doing when expressed alone compared to in the context of virus and whether nsp15 can directly regulate stress granules or acts as a consequence of reducing viral dsRNA levels. Indeed, you indicate that you already have data following up the mechanism of nsp15 action that has not been included. It will also be important to include a more comprehensive discussion about existing knowledge of the function of nsp15 from earlier publications by others.
2. Please include a comparison of the replication dynamics of WT and nsp15 mutant viruses.
3. It will be highly beneficial, although not essential, to compare to nsp15s from other CoVs to determine how conserved this function is. Again, you have indicated you have already made preliminary comparisons.

Minor changes:

1. Quantification of western blots as well as dsRNA levels detected by immunofluorescence should be provided. Please also include the dsRNA dot blot described as data not included to provide additional confidence in the increase in dsRNA levels during replication of the nsp15 mutant virus.
2. Discrepancies in levels of eIF2a-p and PKR-p should be addressed and how this ties in with the proposed mechanism of nsp15 action discussed.
3. Simplify and possibly merge figures where data from other publications is confirmed (make use of supplementary figures) and make clear that these are consistent with existing understanding.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Helena Jane Maier, DPhil

Guest Editor

PLOS Pathogens

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

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: PPATHOGENS-D-20-01225

The manuscript submitted by Gao et al entitled ‘Inhibition of anti-viral granule formation by infectious bronchitis virus nsp15 ensures efficient virus replication’ reports the role of a coronavirus accessory proteins in disabling the stress granules responses.

In recent years several studies have shed light into the role played by stress granules (SGs) assembly in the response to stress stimuli, including viruses, by acting at the crossroads between translation repression, signalling pathways and innate immunity. In turn, many viruses were found to exert inhibitory actions on stress granules assembly. Coronaviruses in particular have previously been shown to manipulate the SG pathways for their replication. For example, MERS 4a protein impairs stress granules assembly while the SARS, SARS-CoV2 and IBV N proteins have all been found to interact with the key stress granules component G3BP1. Recently, Brownsword et al (Viruses, 2020) reported that IBV infection resulted in the assembly of SG in only 20% of infected cells and that IBV prevented the assembly of SG via both eIF2� dependent and independent pathways. The current manuscript investigates further the regulation of stress granules by IBV in mammalian and avian cell lines. The authors confirm previous results of impaired SG assembly in IBV cell with only 20% of cells displaying SG and further identify the accessory protein nsp15 as responsible for this effect. They then demonstrate that the RNAse activity of nsp15 is responsible for this inhibition of the SG pathway. Using a catalytically inactivated nsp15 mutant they shed light on the mechanistic impact of this inhibition showing that nsp15 prevents the assembly of SG that act as signalling hub controlling the IRF3 signalling axis to regulate IFNb production and are sites of innate immunity sensors storage. That part of the manuscript is the most novel and exciting, and bring novel insights into SG function. The experiments are all very convincing and produce both novel understanding of the role of SG during coronavirus infection and how accessory proteins counteract their action.

I have a few reservations/comments that the authors need to bolster their study.

Reviewer #2: Overall, I think this paper proposes an interesting new function of coronavirus nsp15 – or, rather, an interesting consequence of the loss of nsp15 activity.

However, I feel that this central hypothesis is lost amongst a range of largely irrelevant data that raises questions that, actually, may not need to be answered in this paper. For example – nsp15 appears to block chemical induced stimulation of stress granules, but the model says that correct processing of viral RNA prevents the build-up of interferon-inducing dsRNA that, as part of the pathway, would induce stress granule formation. So, how does nsp15 block chemical-induced stress granules? Is this because host dsRNA (or ‘junk RNA’, or some other phrasing) accumulates?

The problem I have, though, is that I am not necessarily sure that it is relevant to the actual central finding of the paper. If only data that showed that shows that “nsp15 activity processes viral RNA so that it no longer induces stress granules” was presented – I would not need to ask that question…. Which is not to say that contradicting data should be hidden (of course) – but, in a sense, does it matter if nsp15 is a ‘multi-functional’ protein, if they can clearly show one function? I don’t think it does.

Reviewer #3: This manuscript from Gao and colleagues describes experiments aimed at determining how the chicken coronavirus infectious bronchitis virus (IBV) inhibits the formation of stress granules. The authors provide extensive data that characterizes IBV infection in a variety of cells types, and that they detect stress granules in a minority of the infected cells (they don’t emphasize the time points they are using in these experiments). Using overexpression studies, they found that expression of nsp15 seems to reduce the number of stress granules that form in response to stimulation with chemicals. To determine if nsp15 is important in the context of virus replication, they generate a nsp15 catalytic mutant of IBV. However, they did not fully characterize this virus or directly compare the replication kinetics of this virus to wild type virus in Vero cells (interferon non-responsive) and interferon responsive cells (such as H1299, a human lung cell line). The authors make the important observation that there is an increase in the accumulation of dsRNA in the nsp15-mutant IBV-infected cells (Figure 7C), but they do not seem to appreciate that the accumulation of dsRNA is likely driving the accumulation of stress granules. The authors are encourage to more carefully consider the recent literature on nsp15 mutant viruses (Kindler et al., 2017 PLoS Pathogens; Deng et al., 2017 PNAS; Deng et al., 2019 J. Virol. and Hackbart et al., 2020 PNAS). All these papers are referenced, but the current work would be significantly improved if the data was generated to directly compare the IBV nsp15 mutant to the published MHV, 229E, and PEDV nsp15 mutants. Overall, the data provided does not fully support the conclusions presented by the authors.

Reviewer #4: Infectious bronchitis virus (IBV) is a coronavirus with avian tropism that causes significant economic losses in the poultry sector. In their manuscript, Gao and colleagues investigate how IBV escapes host antiviral responses. They present compelling evidence that nsp15, a virus-encoded endoribonuclease, is a key enzyme in the suppression of host stress responses, particularly stress granule formation. The authors have developed a nsp15-defective recombinant virus, whose catalytic activity is impaired by the mutation of conserved histidine residues, that has lost its ability to counteract SG formation and induces a strong innate immune response (e.g. IFN-ß and IFIT1 expression). The impairment of SG formation by genetic ablation of two main SG nucleating proteins, G3BP1 and G3BP2, reduced the IFN response to the above-mentioned mutant virus. Further, immunofluorescence studies show the presence in SGs of several molecules of the IFN signalling pathway, including TRAF proteins and the kinases IKK and TBK. Altogether, these results indicate that IBV developed a strategy to evade SGs, which act as a platform that conveys the IFN signalling. This work supports the concept of antiviral SG previously reported, notably in the context of influenza virus and Sendai virus infections.

In general, this study is very comprehensive and well presented. The data is of high-quality. Experiments are well controlled. This manuscript significantly advances our understanding of IBV interactions with the host stress and immune machineries. This study will be a great interest to the field.

**********

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: 1- Using two SG markers the authors conclude that SG are assembled in only 20% of infected cells. To convincingly demonstrate that these foci are genuine SG, they should further provide data showing that these granules are sensitive to cycloheximide treatment, and that cells with SGs are translationally impaired. It is clear from previous studies that G3BP1 or TIA-1 foci can be associated with several types of RNP or RNA granules. In addition, it is clear that SG assembly occurs in a multistep fashion and recent evidence points towards the accumulation of UBAP2L foci seeding larger G3BP1 +ve granules. The authors should address whether in G3BP1 foci -ve cells UBAP2L remains diffused in the cytoplasm or whether it accumulates in foci. This will also help them further refine their final model in the discussion and figure 11. Given their hypothesis that nsp15 may affect the dynamic of SG and their disassembly, the authors should evaluate the impact of nsp15 wt or mt on SG recovery following ARS treatment. If nsp15 promote the disassembly of SG, using sub-acute concentration of ARS would result in differences in kinetics of G3BP1 re-distribution into the cytoplasm.

2- In several figures the authors measure eIF2� phosphorylation levels i.e Figure 2 to start with. In this figure, the authors conclude in the text that IBV does not induce phosphorylation (line 171). However looking at the supporting data on Fig 2 it is clear that at 4 and 8 hpi (H1299) or 12 and 24 hpi (Vero) the phosphorylation is much more intense than in the uninfected controls. The authors should either quantify these western blots or provide a more accurate account of eIF2� phosphorylation by using the quantitative PhosphoTag approach. Later in the manuscript, in figure 3E or 5D the levels (background or infected) of eIF2��seem much lower with no differences between mock/infected. Can the authors explain/discuss these discrepancies? Likewise for PKR-P blots, figure shows a strong induction of P-PKR

Reviewer #2: Now that they have worked out the mechanism, it seems fairly easy to include nsp15 from at least some other coronaviruses (though not necessarily using viral mutants) to help them make the argument that this is pan-coronavirus. It would be even more interesting if MHV/TGEV nsp15s are able to do this but are prevented from doing so in their normal infections.

Perhaps one way of doing this would be to transfect the cells with nsp15s and see if they can 'rescue' the IBV nsp15 mutant.

Reviewer #3: 1. Figure 6. This schematic diagram shows the generation of IBV-nsp15 mutant virus. The kinetics of virus replication of WT and nsp15 mutant should be compared in Vero cells (interferon non-responsive) and H1299 and DF-1 cells (both interferon responsive cells). Studying the kinetics of virus replication and the kinetics of the interferon response, and directly comparing the nsp15 mutant to wild type virus is likely to be important. The immunofluorescence study shown in Fig 6B is only at one time point.

2. Methods, Viral titer determination (lines 631-636). The virus titer of WT and the nsp15 mutant should be determined in Vero cells, not the interferon responsive H1299 cells.

Reviewer #4: 1) The nsp15-deficient virus is a key tool for understanding the strategy by which IBV reduces dsRNA levels and thereby escapes PKR sensing. Although Fig7A provides a Western blot analysis showing that less viral protein is expressed in infected cells, some assays to describe the replication of this mutant virus compared to the wild-type virus (e.g virus production over time analysed by TCID50 assay) should complement this panel. Importantly, the inability of this mutant to degrade viral dsRNA is an essential aspect of nsp15 counteraction mechanism. However, the immunofluorescence panels showing dsRNA levels are poorly convincing (Fig 7C and D). Quantifications should be added. In the same line, the dot blot analysis of dsRNA accumulation in infected cells mentioned in the manuscript (line 277) as “data not shown” should be included in the manuscript.

2) Fig 10 shows the localization of several immune signalling molecules as well as PKR to SG supporting the role of SGs as an antiviral signalling platform. Co-localization measurements for the different proteins (e.g. using Pearson's correlation coefficient using ImageJ or CellProfiler) should be provided to increase confidence in this very interesting observation.

**********

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: >Overall some experiments suggest that nsp15 interfere with the formation of SGs downstream of eIF2� (line 237). However, using the nsp15 mutant the authors convincingly show that nsp15 antagonize PKR activation and eIF2� signalling (figure 6/7) and that nsp15 mt expression rescue eIF2a phosphorylation and SG assembly, and that this leads to IRF3 signalling and IFN production. Thus, it is not quite clear to this reviewer exactly how downstream of eIF2� the block occurs. This section of the discussion may benefit from being clarified.

>the authors seem to completely ignore the 20% of cells that do assemble SGs. The observation warrants a place in the discussion. What are the authors’ suggestion about properties of these cells that would make them susceptible to SG assembly, lower nsp15 expression? Paracrine signalling? Some speculation here would be most welcome.

>within the introduction line 59, the authors limit their introduction to SG to the role of few RBPs and should emphasise as well the role played by RNA aggregation in mediating SG assembly.

>Fig 7A should read eIF2� not eIF2a

Reviewer #2: Figures 1 and 2 (and associated descriptions) can be summarised as ‘we infected cells and nothing happened to stress granules (except late in infection)’. These do not feel worthy of their own sections and descriptions (though, they are important controls for some later experiments).

If this is a common feature of coronavirus nsp15s (which it need not be to have value), why/how do MHV and TGEV induce stress granules? Is there evidence that nsp15 genes are substantially different in these coronaviruses? Either way – this is clearly not a pan-coronavirus mechanism of action (unless, possibly, the expression of nsp15 in MHV/TGEV is much lower than ‘normal’ and is simply not at a high enough level to completely override stimulation of stress-granules by RNA or other means).

The general model from this paper is: without nsp15 endoribonuclease activity, double-stranded viral RNA accumulates in the cell, which triggers stress-granule formation, which stimulates the interferon response, which inhibits virus replication. The other side of that is that nsp15 blocks stress granule formation by the correct processing of viral RNA and preventing the accumulation of dsRNA. Which sounds reasonable. However, the question is: if this is the case, how does nsp15 inhibit the formation of chemically induced stress granules?

If, as suggested, this is a ‘simple’ interaction between nsp15 and RNA – why do MHV and TGEV not do the same thing? They have RNA and nsp15 (unless nsp15 in these viruses is mutated/truncated).

On many bar graphs, the figure legend states the data are “representative of three independent experiments”. Surely the graphs should show the means +/- SD of the independent experiments combined, not the repeated samples of the same experiment. I appreciate that some of these data sets may vary between ‘runs’ and will, therefore, make the graphs look poor, but this can be overcome by comparing to a control or using an alternative plot format (e.g. a dot plot showing all data points).

If statistics are run on three repeats of the same sample, it is hardly surprising that the error will be low and significance obtained. I do acknowledge that in most graphs, based solely on the data presented, the difference is large enough that almost all will be significant anyway, so does not alter any conclusion drawn.

But I have particular concerns about figure 8D. The description appears to suggest (rightly or wrongly) that the authors repeated a titre calculation on the same sample three times (fairly) and then presented that as “representative” of three independent experiments. This is incorrect and, indeed, it is not surprising that this yielded a significant result with an apparent small difference.

Tables 1,2 and 3 should be supplementary data

Figures 4A and 6A seem superfluous.

Figure 11 is too complex and, possibly, unnecessary.

Reviewer #3: 1. Title: does nsp15 inhibit stress granules or does nsp15 inhibit dsRNA formation, which limits the formation of stress granules?

2. Abstract, lines 5-6: Coronaviruses have proof reading activity, so in fact they do not mutant frequently.

3. Abstract, lines 23-25. Do you have direct evidence that nsp15 suppresses the formation of SGs? Or does nsp15 do something else that then results in low levels of stress granules?

4. Introduction. This introduction is about stress granules, but the paper focuses on the role of nsp15. It would be more appropriate if the introduction reviewed the key findings of studies on nsp15 in coronaviruses and arteriviruses, and then pointed out the gap in knowledge in studies of IBV.

5. Methods, line 480. Include that H1299 cells are a human lung cell line that is permissive for IBV replication. Why is a human cell line used for a virus that infects chickens? The species and organ that gave rise to Vero cells (E6 or other?) and DF-1 cells should also be provided here.

6. Discussion. This discussion has many paragraphs that are really background and introduction material about the replication of coronaviruses. It would be more appropriate if the authors focused on a discussion of their findings and directly comparing the results of their study with recent literature.

Reviewer #4: 1) Generally, Western-blot quantifications are missing. Figure 2, are the variations observed in p-eIF2alpha and p-PKR levels significant? PKR levels in panel B give the impression to be induced by IBV infection.

2) The authors state that virus progeny from double knockout G3BP1/2 cell is “more infectious” (line 306). This is not correct. The TCID50 assay only indicates that more virus is released in the supernatant likely because replication is less repressed by the immune response.

3) The role of nsp15 (EndoU) in the evasion from PKR, as well as OAS and RNAse L, was previously reported using a similar approach in the context of the closely related viruses, HCoV and murine hepatitis virus, by Kindler E and colleagues (PLOS Path 2017). While this reviewer is totally enthusiastic about the novelty of the finding concerning the impact on SGs formation (or disassembly in the context of a wild-type infection), the authors should discuss this paper and tune down “the previously unrecognized role of nsp15 in the evasion of PKR” (line 399).

4) P-PKR levels were not measured in Fig 9. PKR activity has been reported by Reineke LC and colleagues (2015) to be enhanced and maintained by SG formation. Could the disruption of SGs by genetic ablation of G3BP1/2 directly influence dsRNA (viral or in polyI:C transfected cells) recognition by PKR?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Figure 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. 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 us at figures@plos.org.

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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Prof Liao,

Thank you very much for submitting your manuscript "Inhibition of Anti-viral Stress Granule Formation by coronavirus endoribonuclease nsp15 Ensures Efficient Virus Replication" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

There‘re basically to points to address. Please provide a better wording or quantification concerning the activation as outlined by reviewer 1, and comment on inclusion, consolidation, or removal of figures 1 and 2 (reviewer 2).

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

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 #1: The authors have addressed the comments I had raised about the previous submission PPATHOGENS-D-20-01225. While they could have done more work to dissect the role of nsp15 on SG recovery the additional data provided elsewhere are sufficient to warrant publication. Some inconsistencies remains in their discussion of eIF2a and PKR phosphorylation that they need to clarify as outlined below.

Reviewer #2: The authors have responded to my main concerns.

Reviewer #3: In this revised manuscript the authors have addressed all the concerns raised by the reviewers. This is an impressive body of work with 13 Figures and supplementary data that supports the conclusions put forth in the manuscript.

Reviewer #4: (No Response)

**********

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: In their revised manuscript, and in their rebuttal to my main point 2 of the original review the authors are now providing quantification of western blots to better discuss eIF2 and PKR phosphorylation patterns. In the updated lines 201-210 the authors write that eIF2s phosphorylation is slightly increased and later that PKR-EIF2-SG pathway is slightly triggered.

The phosphorylation and the pathway are either activated or they are not. Can the authors be more assertive in the text? If their argument is that activation is lower (a measurable) than a control then they should refer to it. i.e directly compare phosphorylation levels to an arsenite or poly(IC) treatment. As it stands the statements are confusing.

Reviewer #2: None

Reviewer #3: (No Response)

Reviewer #4: Gao and colleagues have extensively addressed the reviewers' comments and provided all requested data. With this additional work, my remaining concerns have been addressed and the overall quality of the manuscript quality has greatly improved.

**********

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: Although I feel that the experiments are more complete in this submission.

My main concern in the first round was that the authors were trying to draw broad conclusions about all coronaviruses, rather than focusing on IBV. They have now added in experiments with other coronavirus nsp15s.

My reason for suggesting removal, or consolidation, of figures 1 and 2 was that these feel like a lot of explanation for a relatively minor observation (in context).

This gives this paper a feeling of being very long.

Reviewer #3: none noted

Reviewer #4: (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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you 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. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

Dear Prof Liao,

We are pleased to inform you that your manuscript 'Inhibition of Anti-viral Stress Granule Formation by coronavirus endoribonuclease nsp15 Ensures Efficient Virus 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.

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,

Volker Thiel, Ph.D.

Section Editor

PLOS Pathogens

Volker Thiel

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):