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Review 2: "Release of P-TEFb from the Super Elongation Complex promotes HIV-1 Latency Reversal"

Reviewers noted concerns including the high, toxic concentrations of the inhibitor used, lack of raw data and normalization details, and the need for further mechanistic evidence of the inhibitor's specificity. One reviewer deemed it not informative due to these issues.

Published onApr 09, 2024
Review 2: "Release of P-TEFb from the Super Elongation Complex promotes HIV-1 Latency Reversal"
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Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal
Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal

ABSTRACT The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.AUTHOR SUMMARY Since the start of the HIV pandemic, it is estimated that nearly 86 million people have been infected with the virus, and about 40 million people have died. Modern antiretroviral therapies potently restrict viral replication and prevent the onset of AIDS, saving millions of lives. However, these therapies are not curative due to the persistence of the virus in a silenced or ‘latent’ state in long-lived cells of the body. One proposed strategy to clear this latent reservoir, termed “shock and kill”, is to activate these silenced viruses such that the infected cells can be cleared from the body by the immune system. While several drugs have been developed that can activate latent viruses, none have proven effective at reducing the size of the latent reservoir in patients in clinical trials. Here, we describe a new method for latency reactivation using a small molecule inhibitor of a human protein complex called the Super Elongation Complex (SEC). Inhibiting the SEC enhances viral transcription during active infection and triggers the reactivation of latent viruses, especially when in combination with other latency reversing agents. These results pave the way for developing more effective strategies to reactivate latent viruses towards a functional cure.

RR:C19 Evidence Scale rating by reviewer:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.


Review: This manuscript by Cisneros et al describes the role of the Super Elongation Complex on HIV-1 viral reactivation from latency. In particular, the authors found that members of the SEC complex AFF1/AFF4 may block the interaction of pTEFb and tata and as such, disrupting the interaction between CyclinT1 and AFF1/AAF4 with the small molecule and SEC inhibitor KL-2. They show that KL-2 does not itself reactivate latent HIV but enhances latency reversal in several T cell models of latency and in cells isolated from aviremic participants. The study is of interest to the HIV field but will benefit of some additional experiments to fully demonstrate the role of SEC in HIV latency reversal.  There are some issues that should be addressed before publication.

Major issues:

  • Figure 2.

    • Figure 2C. It is not clear how many times the IP was performed for reproducibility. If more than one was done, quantification of all of them should be provided.

    • Figure 2D. It will be more informative to show % GFP not normalized. Or provide how the normalization was done to evaluate the magnitude of increased in infectivity. Viability of these studies should be provided.

    • Figure 2F. Have the authors look at any other activation markers? In particular CD69 as it is an earlier activation marker than CD25. 

    • Figure 2G-H. The increased in HIV TAR and long LTR could be a consequence of increased viral transcription but also of increased infectivity in general. The data should be normalized to infected number of cells with integrated HIV DNA to ensure that the primary mechanism is increased in transcription.

  • The authors claim that KL-2 works as an LRA enhancer by promoting the release of pTEFb. However, the authors failed to demonstrate that the LRA effects are directly associated with this activity and not an off-target effect of the drug. In particular, as it works as an LRA at levels that it is toxic for the cells and as such, other mechanisms could be playing a role. Further demonstration of the role of AFF1/4 by both knocking out and overexpressing to evaluate whether it phenocopies or blocks the activity will be required to ascertain the specificity of the small molecule. This will be required to fully demonstrate the role of SEC proteins in blocking HIV-1 viral transcription upon latency reversal. 

  • Most data is normalized but how the normalization is done is not described. Raw data as % GFP cells in the JLAT cell lines should be more informative without any type of normalization.

  • The data from PBMCs isolated from ART-suppressed participants is intriguing. The authors should indicate whether the participants were male and female. Although it probably was not done, it will be interesting to compare what is the magnitude of reactivation compared to any other strong stimuli such as CD3/CD28, PMA/Ionomycin or PHA.

Minor issues

  • Line 131. Change healthy to HIV-negative

  • Line 260. Should not be Figure 4A?

  • Line 294. Should not be Figure 4D?

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