Dear Mr. Prabhu,

Thank you very much for submitting your manuscript "Rapid prediction of crucial hotspot interactions for icosahedral viral capsid self-assembly by energy landscape atlasing validated by mutagenesis" for consideration at PLOS Computational Biology.

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, in particular all the comments from reviewer 1.

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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,

David van der Spoel

Associate Editor

PLOS Computational Biology

Arne Elofsson

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In the manuscript “Rapid prediction of crucial hotspot interactions for icosahedral viral capsid self-assembly by energy landscape atlasing validated by mutagenesis”, Wu et al introduce a computational method to predict if mutations in capsid proteins can disrupt viral capsid assembly. There are currently no computational methods to assess this efficiently, and the research is significant. However, there are several major issues in the manuscript that challenged a rigorous scientific assessment of the research. The computational and experimental methods are incomplete, the comparison with experimental results is unclear, and the methodology introduces strong approximations that are not discussed with rigor. Below I listed my specific comments regarding these major issues as well as a number of minor issues.

Major issues:

1) The authors claim that the computational approach is validated experimentally (see, e.g., lines 119–121), but this comparison is unclear in the manuscript. I have a number of questions regarding this issue:

• Were the experiments carried in this research work, or were they published previously?

• Section 3.1 Experimental setup provides limited information about the experiments (lines 437–448). What where the mutations done for each virus and what were the assembly results of these experiments?

• Figures 10 and 11 provide the “validation” of the computational method and experiments, but it was not clear what was the prediction from the computational method, what was the experimental observations, and how they were compared.

2) Some parts of the computational method are described in great detail, but several elements that are key to judge the application of this method to the three viruses studied are missing:

• How were the 3D atomic models of the assembled viruses used to define the discretized Lennard-Jones potentials?

• How sensitive are the results of the computational method to variations to the Lennard-Jones pair potential energy difference?

• How sensitive are the results of the computational method to variations in the criteria of assignment of the Lennard-Jones discrete potential to atom pairs?

3) The methodology assumes a quasi-equilibrium pathway of assembly, where the partial capsids can be mapped onto the final capsid configuration. This is a strong approximation that disregard thermodynamic and kinetic mechanisms established previously

• The limitations of the model should be put in context with the current knowledge of assembly pathways (see for example, Reguera et al Soft Matter 2019, Ning et al Nature Communications 2016, Hagan and Elrad Bioph J 2010, and the references within).

Minor issues

1) Several terms like cruciality, subassembly, or atlasing are used early in the manuscript without having been properly defined. Some of the terms are defined later on in the manuscript, but it takes several pages. The authors should provide definitions for these terms early on in the manuscript.

2) The title of the manuscript claims that is a “rapid prediction” but this claim is based on a vague sentence referring to the computational time needed on a “laptop”. The specs of this laptop should be provided (CPU, RAM, number of processors…) so the community can properly judge this computational time.

3) The authors claim that “Icosahedral capsid self-assembly is poorly understood process” (lines 13 and 14). This is misleading. There are various principles (nucleation process, weak interactions, etc.) that are well established (see references given above). This includes the assumptions used in the computational method presented here. The authors should acknowledge this properly and clarify the aspects that remain open (like the multi-scale aspect).

4) Figure 1a is unclear. In a T=1 each monomer is involved equivalently in each icosahedral axis of symmetry. All proteins are equivalent in such capsid. Do the colors associated to each axis reflect domains in the monomers?

5) Review grammar throughout the text, for example (lines 13-14, lines 44-45, …)

Reviewer #2: The manuscript deals with computational modelling of the assembly of icosahedral viruses. Thereby, interface-scale and capsid-scale modelling approaches are combined. The approach uses the EASAL software of the same group. Overall, the is well written and provides detailed explanations of the complex methods.

The only point is that the validation of the prediction with AAV2, MVM and BMV mutagenesis results. The statement of strong correlation in section 3.3 is not really supported by Figure 11. The authors should elaborate this point more detailed or discuss it more cautiously.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: No: Lennard-Jones discrete force field for each virus studied; mutagenesis experiments

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: 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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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 in PLOS Biology 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, please see http://journals.plos.org/compbiol/s/submission-guidelines#loc-materials-and-methods

Dear Mr. Prabhu,

Thank you very much for submitting your manuscript "Rapid prediction of crucial hotspot interactions for icosahedral viral capsid self-assembly by energy landscape atlasing validated by mutagenesis" for consideration at PLOS Computational Biology. 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.

Please address the comments by referee in the discussion section of the manuscript.

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,

David van der Spoel

Associate Editor

PLOS Computational Biology

Arne Elofsson

Deputy Editor

PLOS Computational Biology

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

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Please address the comments by referee in the discussion section of the manuscript.

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed all the major and minor concerns that I raised in my initial review. I reviewed the manuscript a second time, but I do not have any additional comments.

Regarding the weaker correlation for the BMV case, I would like the authors to consider the following observation: AAV2 and MVM both form and empty capsid followed by the packing of the ssDNA genome. BMV instead co-assembles the capsid with the ssRNA genome, which plays an essential role in coordinating the assembly (Comas-Garcia, Viruses, 11:253, 2019). Empty BMV capsids can assemble in vitro, but the BMV wild-type has selected capsid proteins to co-assemble with the genome. Since the algorithm relies exclusively on information about capsid proteins, the approach seems better suited for viruses that form procapsids rather than viruses that co-assemble with the genome. The information about potential hot spots taking into account co-assembly information is missing in the current approach and might explain the issues the BMV results.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

**********

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

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 in PLOS Biology 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/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Sitharam,

Thank you very much for submitting your manuscript "Rapid prediction of crucial hotspot interactions for icosahedral viral capsid self-assembly by energy landscape atlasing validated by mutagenesis" for consideration at PLOS Computational Biology. 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.

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,

David van der Spoel

Associate Editor

PLOS Computational Biology

Arne Elofsson

Deputy Editor

PLOS Computational Biology

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

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

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 in PLOS Biology 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/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Sitharam,

We are pleased to inform you that your manuscript 'Rapid prediction of crucial hotspot interactions for icosahedral viral capsid self-assembly by energy landscape atlasing validated by mutagenesis' has been provisionally accepted for publication in PLOS Computational Biology.

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 Computational Biology. 

Best regards,

David van der Spoel

Associate Editor

PLOS Computational Biology

Arne Elofsson

Deputy Editor

PLOS Computational Biology

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