Dear Dr. Emerman,

Thank you very much for submitting your manuscript "Highly-potent, synthetic APOBEC3s restrict HIV-1 through deamination-independent mechanisms" 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.

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Sincerely,

Welkin E. Johnson

Associate Editor

PLOS Pathogens

Richard Koup

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 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: In this manuscript, the authors explore combining deaminase domains from individual APOBEC proteins to determine whether this would increase antiviral activity against HIV. The double deaminase domain protein APOBEC3G is the most potent of the APOBECs in restricting HIV, and it is currently unknown whether all of the possible deaminase domain combinations have been evolutionarily explored in nature. The authors determine that combining deaminase domains from APOBEC3C and 3H results in a super-restrictor that limits HIV replication at least as well as or better than APOBEB3G depending on which assay they utilize. They mechanistically determine that the strong antiviral activity is a result of both deaminase activities and enhanced interaction of the novel restriction factor with viral RNA, correlating with increased packaging into virions. Though the practical/translational impacts of the work are unclear, the experiments are very well done, the data are presented clearly, and the results are informative with regard to the evolutionary history and the breadth of evolutionary sampling of possible antiviral APOBECs.

Reviewer #2: In this work the authors demonstrated that a synthetic A3C/A3H di-domain chimera acquired more efficiency in virion packaging and enhanced antiviral activity, which is comparable to the most potent natural di-domain A3G, and the antiviral activity is conferred by both deaminase-dependent and independent mechanisms. The authors also showed that the deaminase-independent activity is correlated to the increased RNA binding affinity of the A3C/A3H chimera. This is a clean work with only minor modifications needed.

1. Figure 4 only shows the anisotropy measurements for A3C-A3H, A3H and A3G. How about the others, e.g., A3H-A3C and A3C?

2. In Figure 5C the No-A3 control has considerable level of G-to-A mutations. Why is it so different from that in Figure 3A?

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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: Figure 2 is representative of 3 experiments. Can the results from the 3 experiments be quantified and included as a bar graph?

The usefulness of the research is not entirely clear. The authors have coined the term “super-restrictor,” and their novel proteins could potentially be useful in future gene therapy approaches to HIV treatment. However, all of the assays are done with HIV-deltaVIF, which is sensitive to APOBEC inhibition. Creating an APOBEC-based super-restrictor that evades VIF inhibition and can thus inhibit WT HIV-1, would be a more exciting advance. Based on published literature regarding the VIF-APOBEC interaction interferface, can the authors create and test such a version of the A3H/A3C constructs?

Reviewer #2: (No Response)

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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: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Emerman,

We are pleased to inform you that your manuscript 'Highly-potent, synthetic APOBEC3s restrict HIV-1 through deamination-independent mechanisms' 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,

Welkin E. Johnson

Associate Editor

PLOS Pathogens

Richard Koup

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 Comments (if any, and for reference):