Dear Dr. Valli,

Thank you very much for submitting your manuscript "Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases" 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. Especially the authors need to address the reviewer 1's major concern, i.e., lack of direct evidence of viral inhibition by ITP/XTP. The reviewer 1 also suggested some feasible experiments. We are expecting that the authors can address it timely using these suggested experiments or alternative experiments the authors deem appropriately.

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.

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[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).

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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,

Weifeng Gu

Guest Editor

PLOS Pathogens

Shou-Wei Ding

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

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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: This manuscript presents a few interesting observations and the authors provided working model, but the conclusions remain largely descriptive without plant host direct genetic evidences. Without evidences based on these mutant plant materials invovled in ITP and XTP biosynthesis, the authors claim a not directly demonstrated conception about UCBSV requires HAM1 to infect Cassava due to elevated levels of non-canonical nucleotides. Second, there is no virus infectivity difference for these UCBSV-HAM1 mutants. Therefore there is no direct relationship between its enzyme activity (HAM1) with virus infection.

Reviewer #2: In this manuscript, using an infectious cDNA clone and reverse genetics approach, the authors found that the Ugandan cassava brown streak virus (UCBSV)-encoding Maf/ham1-like protein is functional when covalently linked to the viral RdRP for adaptation to its natural host plant cassava, in which over-accumulation of non-canonical nucleotides (ITP/XTP) was detected by HPLCMS/MS experiments. This study is novel and provides exciting insights into the evolutionary biology of plant viruses, under the high concentration of non-canonical nucleotides pressure inside the host, by incorporating an ITP/XTP pyrophosphatase into RdRP to support successful replication and infection.

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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: 1. In the response letter page 1, the author claimed that "These results are the first ones supporting the idea

that host non-canonical nucleotides play a role in antiviral defense, something considered by other scientists

but not yet published (e.g., https://www.gu.se/om-universitetet/hittaperson/martinlagging

“inhibition of ITPase may point to novel antiviral and antibacterial strategies”). Again the authors claimed that this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava but lower ITP and XTP levels in Nicotiana benthamiana. Could you please provide the original set of data for each of 12 samples? Why no statitic analysis for Fig. 4? Also some host genetic manipulation experiments could be provided more direct evidences on this points by silencing some genes invovled in ITP and XTP biosynthesis. Also with these plant genetic manipulation data such as VIGS or CRISPR/CAS9 genome editing will provide a direct evidence to show it is a novel host target for antivirals.

Reviewer #2: The conclusions are appropriate and well supported by the experiments. This reviewer also appreciate that authors have made efforts to address issues in their revision.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. please add Potyvirues before RNA-dependent RNA polymerases in the title.

2. please remove the words such as “Ebola of plants” in the abstract.

3. In the abstract and main text of the manuscript, please consider to replace the word "unexpected high concentration in non-canonical nucleotides in cassava", which is

Reviewer #2: (No Response)

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Dear Dr. Valli,

We are pleased to inform you that your manuscript 'Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases' 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,

Weifeng Gu

Guest Editor

PLOS Pathogens

Shou-Wei Ding

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 my concerns on the last version of manuscript. No further suggest here.

Reviewer #2: (No Response)

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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: N.A.

Reviewer #2: All the concerns have been addressed and discussed. I have no more criticisms.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: N.A.

Reviewer #2: (No Response)

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Reviewer #1: Yes: JIAN YE

Reviewer #2: No