HIV-1 accessory protein Vpr possesses a cryptic p300-dependent transcription-promoting activity that is blocked by histone deacetylases in CD4+ T cells.

PLOS Pathogens

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Editor-in-Chief

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Reviewers' Comments:

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: Lewis et al. assess the role of HIV-1 Vpr and HDAC inhibition in latent reservoir formation and HIV-1 promoter activity. They tested a series of proviral constructs in primary CD4+ T cells and determined that Vpr promotes LTR activity in the presence of the HDAC inhibitor Vorinostat. Further examination of this phenomenon suggested that Vpr promotes a central memory phenotype and that these cells are more resistant to Vpr-induced apoptosis. While several of the experimental observations herein confirm previous observations from prior studies, the novelty comes from the extensive use of primary CD4+ T cells. I have some concerns regarding the interpretation of some of the results and the magnitude of the changes induced by Vpr. Questions and concerns are detailed below.

Reviewer #2: Lewis et al explores activities of HIV-1 Vpr in CD4+ T cells with a focus of on whether Vpr influences HIV-1 transcription and the establishment of latency. Key findings are that Vpr and HDACs may interact to repress HIV transcription, and this activity is dependent on p300. In addition, Vpr influences primary CD4+ T cell survival and an expansion of Tcm cells, which have been shown to harbor latent proviruses. Strengths of the paper are the general approach of utilizing primary CD4+ T cells, the use of HIV-1 clones with a single accessory protein and utilizing Vpr mutations to map domains to specific functions that suggest Vpr may be through multiple mechanisms influencing HIV replication in primary CD4+ T cells. However, the potential multiple activities of Vpr, clouds the primary message of the paper which tries to emphasize the Vpr-HDAC-p300 axis. There are also some concerns as to the modest data and lack of exploring and discussing direct and indirect mechanisms of Vpr and HIV transcription. Specific comments are below.

The data in which Vpr+Vorinstat, a HDACi, examines how HDACs might alter HIV-1 infection, transcription, and latency show about a two-fold difference in HIV-1 expression, as monitored by GFP MFI and HIV-1 transcription. There is no discussion whether these differences are significant.

It is difficult to know if Vpr+Vor reflects difference in transitioning to latency since no experiments are performed to look at the ability to reactivate proviruses after the extended culturing.

It is stated that the percentage or number of cells that are expressing HIV do not significantly change, however, the flow cytometry profiles for the different experiments and different viruses suggest a great deal of variation between the different viruses and experiments. Does this reflect different fitness between viruses, experimental variation, or some other variable?

The use of different HDAC inhibitors would be interesting in that it might address potential off-targets of Vor, and provide insights into the role of different classes of HDACs.

The data with the trans expression of Vpr seemed modest and at times contradictory to the data obtained with the viruses. Are these significant differences?

The impact of Vpr on Tcm is interesting. It would be informative if they examined if this could lead to more latently infected cells by trying to reverse latency.

Has differential regulation of different HDACs been examined in T cell subset? This should at least be discussed in greater detail.

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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: - Why are the percent positive cells so different between the Thy1.2 and eGFP markers for the flow results? Based on the system, I would have anticipated that percent positive for eGFP would be lower, or at least equivalent, to percent Thy1.2 positive cells. For example, in Figure 1C 63% of the cells are eGFP positive while only 20% are Thy1.2 positive? This changes to 92% eGFP positive and 63% Thy1.2 positive after Vorinostat treatment.

- The bar graphs throughout are a little misleading for assessing the contribution of Vpr to reactivation of latent reservoirs with vorinostat. In general, Vpr expressing cells activate at ~10-fold lower frequency compared to control cells (ex., Figure 1C control is 18% whereas Vpr cells 1.13%; Figure 3C control is 15.1% while Vpr is 0.62%; Figure 4 control is %6.52 while Vpr is 0.23%). The bar graphs suggest those Vpr infected cells contain brighter eGFP expression, but the flow plots don’t seem to indicate that. Instead, the increase in MFI seems to be due to a smaller denominator, which may not be biologically relevant.

- The authors should validate that the increase in eGFP MFI in Vpr+vor cells translates to detectable increases in viral protein production compared to control+vor treated cells. This is particularly important given the discrepancy between the flow plots and bar graphs discussed above.

- It is odd that several of the Vpr mutants show significantly diminished protein abundance when expressed in cis compared to expression in trans, and that the eGFP MFIs were relatively equivalent. Did the authors confirm that the percent of cells that form latent reservoirs with these new viruses were relatively equivalent compared to wild-type?

- Because several previous studies have suggested that Vpr recruits p300 to the LTR to enhance transcription [PMIDs: 9560267, 12379213, 10505122], I would have anticipated that Vorinostat + p300 inhibition would block the increase in eGFP MFI. The data in the main figures in conjunction with the data presented in Figure S6 suggest this is not the case.

- Figure 4A indicates a strong increase in CD45A+-CCR7+ cells in Vpr infected cells compared to control cells, but there are significantly fewer Vpr infected cells in the CD45A+-CCR7+ flow plot. Could this increase be due to sampling bias? In other words, if the events were normalized between the plots would the increase observed in the Vpr samples be more comparable to control? For example, the total events in Figure 4B are more similar and the increase in CD45A+-CCR7+ cells in the Vpr infected cells is negligible compared to control cells.

Reviewer #2: No key experiments. Using different HDAC inhibitors would be informative.

Experiments that directly address latency would enhance the significance of findings.

In addition, demonstrating Vpr and p300 complexes at the LTR would strengthen the model.

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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: - I’m not sure if it an upload issue or a conversion issue but many of the figures are blurry and the legends are too difficult to read. This is particularly important for trying to interpret the flow cytometry data when comparing percentages across populations.

- Percentage label in Vpr trans mCh experiment in Figure 2D is offset to the right overlapping the other flow plot

- S5A labels are shifted

Reviewer #2: Minor comment: Potential indirect effects on HIV transcription should be included on the figure 6 model. Plus, I am not convinced their data support a mechanisms of Vpr+Vor directly influences LTR activity.

Minor comment: ref 21 and 23 are the same?

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Reviewer #1: No

Reviewer #2: No

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Reproducibility:

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Dear Ms. Lewis,

We are pleased to inform you that your manuscript 'HIV-1 accessory protein Vpr possesses a cryptic p300-dependent transcription-promoting activity that is blocked by histone deacetylases in CD4+ T cells.' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

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Academic Editor

PLOS Pathogens

Richard Koup

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

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Michael Malim

Editor-in-Chief

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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: The authors have adequately addressed my previous comments/concerns

Reviewer #2: The revised manuscript addressed most of my concerns including better statistical explanations, additional experiments that address mechanism, and expanded discussion.

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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: The authors have adequately addressed my previous comments/concerns

Reviewer #2: No major issues

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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: The authors have adequately addressed my previous comments/concerns

Reviewer #2: None

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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.

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Reviewer #1: No

Reviewer #2: No