PONE-D-20-39293

Identification of Celastrol as a novel HIV-1 Latency  Reversal Agent by an Image-Based Screen

PLOS ONE

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: In this publication by Liu et al, has found a new class of HIV-1 Latency Reversing Agent (LRA), namely celastrol, following the screening of a natural compound library. The authors very convincingly demonstrated that celastrol quite selectively stimulates latently infected T cells, but not much to the uninfected resting primary CD4+ T cells. However, the precise underlying mechanism through which celastrol works is under investigation. The Rice lab has contributed enormously to the field of HIV transcription and latency, by doing a number of seminal studies. This publication marks the first set of results, which are very exciting and groundbreaking. In this investigation, for disrupting as minimal as possible the cellular state during experimentation, authors used a novel image-based assay.

Overall, this is a very well controlled and organized ongoing study. In this manuscript authors have presented their initial results. Authors convincingly demonstrate the strong synergistic activity of celastrol with other LRAs, however, defining of the underlying molecular mechanism is still under investigation.

Major Concern:

• Given the fact that celastrol synergizes with other LRAs in reactivating latent HIV, it will be interesting to check if celastrol enhances the cell/nuclear translocation of drugs/factors.

• Interestingly, celastrol inhibits NF-kB, and reduces Cyclin T1 levels, but still able to enhance HIV transcription, indeed directing towards a novel mechanism for HIV transcription. I will be crucial to show if celastrol also enhance HIV replication in your system.

Minor Concern:

• Please clearly indicate which NF-kB (p65) residue was assessed for determining the NF-kB phosphorylation.

• Line 75 of page-5 needs editing, maybe they want to say” An important property of any LRA is the absence of significant resting T cell activation, an activity that may induce systemic inflammation”.

• Line 294 of page-14 needs editing, maybe authors want to add inhibition in the sentence “Additionally, celastrol has been reported to INHIBIT HIV-1 Tat activation of viral gene expression directed by the viral LTR in the U937 cell line [35]”.

Reviewer #2: The current manuscript from Rice and colleagues titled “Identification of Celastrol as a novel HIV-1 Latency Reversal Agent by an Image- Based Screen” describes a new and novel compound that may reverse HIV gene expression along with other LRAs. Here the authors used an image-based assay for compounds that minimally activate primary CD4+ T cells isolated from healthy donors and screened a natural compound library and identified celastrol, a pentacyclic terpenoid, as a novel LRA. Celastrol induced a limited amount of aggregation of CD4+ T cells (Low CD25 and CD69 induction) from both donors and functioned additively with JQ1, vorinostat, romidepsin, and bryostatin. Interestingly, Cyclin T1 was strongly down-regulated when adding celastrol in Jurkat 2D10 cells. Overall, the manuscript looks interesting but rather incomplete. It is not clear what is the mechanism of activation for Celastrol, since it decreases phospho-NFKB in both cell lines and donor samples. Also, I am not convinced that the combination drug treatments are not toxic over time. In fact, if the cells are starting to die in 24-48 hrs, that would explain why the virus gets activated and gets out of the cells. Finally, the authors should look for presence of short TAR RNA in their treated samples which can soak up the Tat from the system.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments-Liu et. al.pdf

Identification of Celastrol as a novel HIV-1 Latency  Reversal Agent by an Image-Based Screen

PONE-D-20-39293R1

Dear Dr. Rice,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yuntao Wu

Academic Editor

PLOS ONE

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