Dear Dr. Svensson:

Thank you very much for submitting your manuscript "Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells" (PPATHOGENS-D-19-01861) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

David T. Evans

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Both reviewers felt that the revised manuscript addressed most of their previous concerns and is substantially improved, but raised a few remaining questions. Please address these remaining points as best you can given the limitations of your model system.

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: PLOS Pathogens

Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells (Birgitta Lindqvist, Sara Svensson Akusjärvi, Anders Sönnerborg, Marios Dimitiou, J. Peter Svensson) (PPATHOGENS-D-19-01861)

The authors added comments, corrected or performed additional experiments to address the majority of the reviewers concerns. However, some points are still not convincing and need to be further addressed.

Reviewer #2: Overall, it seems to me that the authors have addressed most of the questions raised by reviewers, though some caveats in this study remain and could be discussed.

The major caveats of the primary cell latency model include a low basal level of GFP+ cells and low induction suggested by GFP+ cell rate, which was pointed out by reviewers in the major suggestions. These caveats are in the nature of the bcl-2 primary cell model. They were well addressed by the authors by revising the text and explained the data more extensively. Increasing the number of donors and substantial statistical analysis might also have helped to clarify the theory. However, it’s noteworthy that the caveats associated with the model do significantly weaken the conclusions. The initial frequency of infected cells was around 10% (line 218.) After weeks of culturing, GFP+ rates decreased to 1%, which was lower than 2% even upon stimulation. It suggests that 80-90% of HIV provirus were resistant to the reactivation, which should be kept in mind when interpreting the data in figures 5 and 6.

I doubt that switching between Jlat and bcl2 model (especially in figure 5-7) added any value to the study other than technical convenience. Epigenetic character is heavily system dependent. I don’t think that the data using primary cells and cell lines are comparable or interchangeable.

Also, I understand epigenetic is the main focus of this study. But, other cellular factors likely contribute to the transcriptional status of provirus together with epigenetic factors, which should at least be discussed.

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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: Major comments:

1) In this work, the key conclusion that the proviruses actively transcribed following T-cell activation bear enhancer chromatin marks is based on the loss of this epigenetic mark and on a comparison with ChIP seq data from cellular genes. The reviewer 1 asked for additional experiments and controls to improve this conclusion. To address to this point, the authors used the GNE049 drug which depletes H3K27ac specifically at enhancers in 5A8 cells which have been described in Figure 8 to contain low level of H3K27ac before activation. The 5A8 cells are not the best model to conclude on the need to lose enhancer marks to reactivate HIV from latency since 5A8 cells contain high level of H3K27Ac in the provirus after PMA activation. It is therefore not surprising that increasing doses of GNE049 decreased the percentage of GFP intensity (Fig. 7D). To improve their experiments using the GNE049 drug, the authors should use a better HIV-1 latency model (in this case HIV-1 latently-infected cells with higher amount of H3K27ac enhancer mark) coupled to viral transcripts measurement.

2) No explanation is given by the authors regarding the levels of negative epigenetic marks that remained constant following TCR activation (Figure 7A). The authors responded by stating : “that heterochromatin marks at the provirus are not affected by T-cell activation as an indication that latency of these proviruses are not reversed, at least not to a level that we can reliably measure it in this experiment”. The authors should further discussed (in regard to previous work in the literature) the epigenetic marks H3K9me3 and H3K27me3 which remained constant after TCR activation and provide the percentage of GFP positive cells in absence and presence of TCR activation, supporting the absence of HIV-1 latency reversal.

Reviewer #2: no

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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: Minor comments:

1) Reviewer 1 mentioned that in Fig 2F: at 3dpi, a detection of all HIV-1 transcripts should be observed. However, no detection of these transcripts was observed. In Figure 2F, all HIV-1 transcripts were not observed with no detection of transcripts downstream nuc1. The authors responded to this point by stating :” This discrepancy might be due to technical issues or that transcription at 3 dpi is strongly reduced and the observed protein levels are lingering from earlier translation”. They also included a section in the Discussion on the cellular viability issue. The authors should provide information and/or additional figure regarding the cell viability. Same remark for Figure 3C at 70 days post-infection

2) Reviewer 3 observed variations throughout the proviral genome without statistical analysis of the significance of these variations over the time post-infection (Figure 5A). The authors answered by performing statistical analysis on the dataset. However, they observed statistical significance of heterochromatin accumulation at several points. Thus, the authors should be careful when stating (line 314): “The constitutive H3K9me3-mark appeared to be uniformly distributed over the proviral body by 30 dpi” and should further discuss these variations.

3) The authors should explain and discuss the contradictory results regarding the H3K27ac mark obtained in 5A8 and HIV-1 BCL2 cells (Figures 6E and 7A).

Reviewer #2: please see above

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Reviewer #1: No

Reviewer #2: No

Dear Dr. Svensson,

We are pleased to inform that your manuscript, "Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells", has been editorially accepted for publication at PLOS Pathogens. 

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Best regards,

David T. Evans

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer Comments (if any, and for reference):