Dear Dr. Wang,

Thank you very much for submitting your manuscript "Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS." 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.

The reviewers agreed that the manuscript tackles an interesting and important aspect of KSHV by seeking to define a molecular determinant for gender disparity in KS pathogenesis. That said, there were numerous concerns that will require additional experimentation to support the conclusions of their findings. The reviewers point out the need for additional controls in experiments (antibodies for ChIP, genetically matched cell lines, reporter constructs, knockdown efficiency). They also noted disparate results between assays (mRNA and protein, microscopy and IFA) and the need for careful interpretation of data using the inducible cell lines. Lastly, statistical analyses were lacking and careful editing are needed to improve clarity throughout the paper.

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.

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,

Laurie T Krug, PhD

Associate Editor

PLOS Pathogens

Blossom Damania

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

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

The reviewers agreed that the manuscript tackles an interesting and important aspect of KSHV by seeking to define a molecular determinant for gender disparity in KS pathogenesis. That said, there were numerous concerns that will require additional experimentation to support the conclusions of their findings. The reviewers point out the need for additional controls in experiments (antibodies for ChIP, genetically matched cell lines, reporter constructs, knockdown efficiency). They also noted disparate results between assays (mRNA and protein, microscopy and IFA) and the need for careful interpretation of data using the inducible cell lines. Lastly, statistical analyses were lacking and careful editing are needed to improve clarity throughout the paper.

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 manuscript by Ding et al. Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS addresses the molecular mechanism for why there are more cases of KS in males compared to females. The authors focus on the androgen receptor (AR) and demonstrate the ability of AR to upregulate lytic gene expression, especially PAN RNA. While this is an interesting study which attempts to address the large gender disparity in the development of KS, overall some of the data isn’t convincing and in some places the writing is unclear or confusing.

Reviewer #2: This report by Mingzhu Ding et al., describes “androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS”. Using anti-AR ChIP analyses, the authors found a region in the PAN promoter binding AR. This binding appears important for KSHV activation and cell migration (infiltration). The story will be more attractive if the authors could extract their data more carefully and have a deletion or mutation control of the identified AR-binding site in the PAN promoter-Luc reporter assays.

Reviewer #3: In the manuscript entitled “Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS” the authors attempts to corelate the relative higher abundance of the KSHV infection in males with the presence of the androgen receptor and the effect of ligand binding to it. The study provides new information about how the androgen receptor is acting as a transcription factor promoting transcription of some of lytic genes including RTA. The study addresses a central question related to KSHV and provided data that has strongly supported their conclusions. There are few minor queries that needs to be addressed.

1. The authors showed the expression of the androgen receptor (AR) transcripts in the B-cells and also by western blot. The protein atlas data clearly showed that AR is detected at the mRNA levels in B cells but not at the protein level. How do the authors address this discrepancy? I agree that KSHV infection may increase AR expression but detecting them at the protein level in such high intensity raises my concern. Is the specificity of the antibody carefully validated?

2. In Figure 2A, B the authors showed that BJAB and DH75 which are KSHV negative Burkitt’s cell lines express almost undetectable levels of AR, while BC and BCBL both KSHV positive cells have a very strong expression pattern. BCBL in presence of the DHT (AR ligand) has increased expression of the AR. The authors may want to compare cells with isogenic background like BJAB and BJAB-KSHV, or primary B cells infected with KSHV. This will support their conclusions that KSHV is responsible for increased AR expression. The varying cell background can be problematic in that they may have genetically diverse changes which can make interpretation of the results a bit difficult with high confidence.

3. The microscopic image of BJAB and BCBL in presence or absence of the DHT shows enhanced expression of the AR in BCBL treated with DHT compared to the BJAB counterpart. However, comparing this data with the western blot (Fig2B) seems a bit contradictory. In the WB the BJAB in presence or absence of the DHT has no detectable expression of the AR but by microscopy they can detect some signal. The fold change of expression in BJAB and BCBL1 in WB and by microscopy is difficult to correlate and needs some explanation. The authors should double check this and provide statistical analysis of these data which will be more convincing.

4. The authors mentioned that lentiviral overexpression of the AR itself can induce the lytic reactivation even in the absence of the DHT treatment. The authors explained this finding based on previous published studies where they showed that AR can form complexes with chaperons, HSPs and immunophilins, and gets transported from the cytosol to nucleus. The authors can expand with more emphasis on how this may function in nuclear localization and subsequent viral activation.

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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: In the introduction, it’s unclear if AR, which is a membrane-associated receptor, is always present in the nucleus to exert functions as a transcription factor or if this function of AR is dependent on additional factors. Is AR and the localization dependent on KSHV infection?

Figure 3. The ChIP-seq experiments are difficult to interpret because the IgG controls are not included in figure and according to the materials and methods “the IgG group was not used as a control for its strong bias”. It’s unclear what is the IgG bias and what control was used in it's place. Also it is not stated in the text if the cells used for ChIP-seq are latent or induced iSLK.219 cells. The treatment of the cells is “iSLK.219 cells were pretreated with 10uM DHT or its solvent control MeOH for 48 h”, does this mean that the solvent control was used as the ChIP control instead of the IgG? The authors should include an image of ChIP peaks from the solvent control AR-IP with the DHT AR-IP to determine the difference in AR-IP with and without DHT treatment.

Figure 5. The description of the experiments in the text and figure legend was difficult to follow and the interpretation of the results was unclear.

Figure 6. In the luciferase assay the authors show the promoter activity from samples that have either the RTA expression plasmid (RTA-pCMV-HA) or the AR expression plasmid (AR-pCDH-sf3). It would be informative to include the promoter activity from samples that have RTA and AR expressed together since that is relevant during reactivation.

Figure 7. How do the authors know that the changes in RTA expression (panel b, e, g and h) are from the endogenous viral promoter, which they state is regulated in part by AR, and not the doxycycline-inducible RTA that should not be affected by AR?

Figure 8. The description in the text concerning the results of the iSLK.219 cell invasion is “PAN RNA is necessary for AR-mediated oncogenesis”, as iSLK are already transformed cell it is inaccurate to state these experiments demonstrate AR-mediated oncogenesis. Yes, the results demonstrate AR contributes to cell invasion but these experiments do not demonstrate oncogenesis.

Reviewer #2: The story will be more attractive if the authors could extract their data more carefully and have a deletion or mutation control of the identified AR-binding site in the PAN promoter-Luc reporter assays. Here below are more specifics:

1. Introduction: line 84-85, the cited reference #21 is not appropriated and should go with the original publications in 1996 by Sun R., et al. and Zhong W, et al. I don’t know how reliable the data in the cited reference #25 is due to not appropriate controls and should be removed.

Fig. 2. Please delete the figure 2C. Since BJAB cells don’t express any AR in the panel 2a and 2b, fig. 2c for BJAB AR staining must be non-specific.

Fig. 4. Not sure with the data in figure 4d for RTA AR-binding regions -293-154 and -325- 202 as their binding affinity was ten-fold lower than other two. In addition, the binding ratio in 4d is inconsistent with the gel data in 4e where the RTA1 and 4 had strong binding to AR in the 4d, but less binding in the 4e in agarose gel analysis. Moreover, the figure 3a does not show a ChIP peak for AR binding to these RTA regions and thus conflicts with the data in panels d and e. Why don’t you map the AR binding region (~200 bps) to a smaller region more precisely in the PAN promoter, instead? Please spell out PSA.

Fig. 5. It appears the full-length wt AR was never used in the EMSA binding assay, although both truncated forms of AR, in particular the DBD domain-containing short version of AR, displayed a strong binding. However, the data in panel e are problematic and should be deleted due to formation of the protein-probe aggregates that never got into the gel! There is no gel-shift even for the two truncated AR in the absence of any “cold” competitors.

Fig. 6. For the panel 6a, ORF57 binds to the 5’ end of PAN RNA (Massimelli M, et al, IJBS, 2011; JVI 2013), but not to the promoter DNA region. For PAN promoter, Massimelli et al, showed that RTA display only a small activity in the absence of ORF57 (for RNA stability). I am not sure if anyone showed ORF57 specifically and directly binds to the PAN promoter DNA. By including RTA promoter for the luciferase assay, you will need to provide a better evidence why RTA promoter should be included because your data from Fig. 3a conflicts with the data in Fig. 4. Panel 6b Western blot does not have an equal amount of AR expression for every promoter tested in the assays. Panel 6d bar graphs were not aligned well with all individual proteins used in the study. Protein markers should be shown clearly in all WB panels. Why was a pORF57 reporter included in the study? Does ORF57 promoter have AR binding sites? At least, l could not see any ChIP results from this region from Fig. 3. Surprisingly, the ORF57 promoter responded to you AR! A minus AR-binding region in the PAN promoter in the Luc-reporter should be included as a control in Fig. 6.

Fig. 7. I don’t think the panels a-c are necessary for your study because it was under Dox induction. Most exciting results are reactivation of KSHV lytic infection by ectopic expression of AR lentiviruses. Where is the data showing the knockdown efficiency of siAR? Was the KD of AR affecting cell viability or growth?

Fig. 8. Data in fig. 8b conflict with the data in fig. 8a and the quantitative data in the panel b did not support your observations in the panel a. The same problems for the panels c and d. Data in the panel e are acceptable. The question is what was the knockdown efficiency of siPAN? I am assuming the KD efficiency would be low as the PAN is so abundant.

Reviewer #3: There are 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: Page 2, line 26: “preferentially infects”, while there is a clear evidence demonstrating that KS predominately occurs in males, the line “preferentially infects” suggests that the susceptibility between males and females is also different which has not been documented. The authors may want to reword this sentence for accuracy.

Page 2, line 45: “prokaryotic noncoding RNA”, unclear what the authors are attempting to refer to, maybe PAN RNA, but “prokaryotic” is not an accurate word in this sentence.

Page 3, line 83-84: “In lytic or abortive lytic-infected cells, the entire viral genomic transcription cascade occurred”, this isn’t completely accurate as during an abortive lytic infection it can fail to proceed at different points during reactivation without the entire transcriptional cascade occurring.

Page 7, line 188: Which secondary antibodies were used and what is the manufacturer?

Page 8, line 237-238: “Because of the smaller size of the KSHV genome, the IgG group was not used as a control for its strong bias”, if this is the case what was used as a control for non-specific binding?

Page 10, line 288: “KSHV DNA was extracted following the manufacturer’s protocol”, what was the kit and manufacturer being used?

Page 11, line 306: What is the concentration of doxycycline used for induction?

S1 Table: In the section with qRT-PCR primers “qRT-TMPRSS2 F” is listed twice and it is the exact same sequence. It is unclear from the main text in which experiment TMPRSS2 was used in qRT-PCR assays.

Reviewer #2: 1. Discussion was not in a good shape and should address various issues from the results.

2. It would be better to have someone good in English to proofread the entire manuscript before submission.

Reviewer #3: See review

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Erle S. Robertson

Figure Files:

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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Wang,

Thank you very much for submitting your manuscript "Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS." 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.

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,

Laurie T Krug, PhD

Associate Editor

PLOS Pathogens

Blossom Damania

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 #2: No

**********

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 #2: No

**********

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 #2: Authors addressed most of my comments in this revision and the revised manuscript is now in much better shape. However, the author defended that ORF57 binds to PAN promoter DNA by citing a paper back to 2007 by JVI where the authors in JVI publication even realized that “it is unclear whether direct binding of Mta to Rta contributes to our ability to ChIP Mta on the nut-1/PAN promoter in vivo” (p13310, JVI 81: 13299-13314, 2007) in addition to miss a non-specific DNA control in their in vitro PAN promoter DNA binding assays with his-tagged ORF57. Thus, the diagram in Fig. 5a should be revised by deletion of the ORF57-binding sites both upstream and downstream of the PAN RNA transcription start site. As an RNA-binding protein, one of major functions of KSHV ORF57 is to stabilize PAN RNA and other target RNAs.

**********

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 #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.

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 see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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. Wang,

We are pleased to inform you that your manuscript 'Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS.' 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.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Laurie T Krug, PhD

Associate Editor

PLOS Pathogens

Blossom Damania

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):