Dear Dr. McCormick,

Thank you very much for submitting your manuscript "KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication" (PPATHOGENS-D-19-01170) 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 problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.

Sincerely,

Sankar Swaminathan, MD

Associate Editor

PLOS Pathogens

Shou-Jiang Gao

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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While the reviewers appreciated the thorough responses to the prior review, it was pointed out that the new experiments did not confirm the observed UPR related effects in another cell type. The reviewer points out that it is not clear whether the UPR effects are a virus-specific phenomenon and it cannot be concluded that this is a cell dependent variability. It would seem reasonable to attempt to show whether this is a virus-strain or cell based difference. While it might be more difficult to generate a B lymphocyte cell line with the recombinant virus, it should be possible to examine the phenotype of the recombinant virus in another epithelial cell line.

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 revised manuscript Johnston & McCormick characterize the kinetics of UPR activation during KSHV reactivation in iSLK and Trex-BCBL1 cells. They nicely show activation of UPR as evidenced by ATF6 cleavage, PERK phosphorylation, and Xbp1 splicing, but reveal that subsequent UPR-triggered transcriptional responses and XBP1s protein accumulation do not occur. Unexpectedly, overexpression of XBP1s appears to have opposing outcomes in iSLK versus the BCBL1 model, but knockdown or pharmacological inhibition of UPR prior to reactivation suppresses late stage events of the viral lifecycle in both cell types. This leads them to hypothesize that components of the UPR are being repurposed for viral benefit. Although the mechanism by which outputs of the UPR are being modulated by the virus remains unknown, this study nicely characterizes the UPR activation landscape during KSHV replication. The resubmission is improved through inclusion of data suggesting that the blunting of downstream UPR responses is not due to host shutoff and through new data bolstering the conclusion that KSHV inhibits the ISR response.

Reviewer #2: In this revision of their manuscript the authors have addressed the majority of the points raised by the reviewers. However, in addressing the issue that they only show inhibition of KSHV replication by XBP1s in iSLK cells they found that this did not hold true in the BCBL-1 derived cells. They conclude that this is due to cell type differences. However, two issues with this conclusion arise. First, the iSLK.219 virus is derived from JSC-1 cells and is heavily modified through introduction of multiple changes to the viral genome while the BCBL-1 derived virus is from a different primary effusion lymphoma sample that does not reactivate as well as the JSC-1 derived virus and is not a modified recombinant virus. Therefore, the differences in the ability of XBP1s to inhibit replication could be virus strain specific or due to the engineered alterations to the viral genome rather than cell type specificity. Secondly, the effect of XBP1s does not occur in the more relevant B-cell only in the highly transformed iSLK cells. Therefore, both viruses should be used in a relevant cell type to determine the importance of the inhibition of replication by XBP1s.

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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: none

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: none

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

Dear Dr. McCormick,

We are pleased to inform that your manuscript, "KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication", has been editorially accepted for publication at PLOS Pathogens. 

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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,

Sankar Swaminathan, MD

Associate Editor

PLOS Pathogens

Shou-Jiang Gao

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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The response and revised manuscript address all the questions raised by the reviewers.

Reviewer Comments (if any, and for reference):