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Review 3: "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 3: "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
Description

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:

Not informative. The flaws in the data and methods in this study are sufficiently serious that they do not substantially justify the claims made. It is not possible to say whether the results and conclusions would match that of the hypothetical ideal study. The study should not be considered as evidence by decision-makers.

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Review: HIV remains uncurable due to the presence of latently infected T cells that persist despite antiretroviral therapy. One of the main strategies to eliminate latently is to reactivate the latently infected cells using drug compounds. Cisneros et al describe a potential new drug compound that can facilitate HIV latency reversal. This drug targets the Super Elongation Complex (SEC) to release positive elongation factor b (P-TEFb) from negative regulatory complexes. Once released, P-TEFb can be recruited to sites of proviral transcription and reverse HIV latency. P-TEFb can be sequestered with numerous regulatory proteins. However, the release of P-TEFb from the SEC has not been thoroughly explored. Cisneros et al aim to explore the impact of P-TEFb release from SEC and utilises both in vitro cell lines and primary CD4+ T cells and ex vivo CD4+ T cells to make their conclusions. The authors have found that SEC is not required for HIV replication and that combining the SEC with other latency reversing agents such as JQ1 reactivate latent HIV in both cell lines and PBMCs from people living with HIV.

Although the study follows a logical order in the aim to investigate both SEC and its importance in HIV latency reversal as well as the SEC inhibitor KL-2, there are a few concerns with the study. First and foremost is that many of the conclusions made in the manuscript are based on only one experiment and statistical analysis is completed on technical replicates. Conducting experiments only once reduces the reproducibility of the scientific research, and repeating experiments can confirm the reliability of the initial observations. In the instance where more than one biological replicate has been completed, statistical analysis was still performed on technical replicates. Moreover, conclusions on n=1 reduce the statistical power of the analysis. The choice in statistical testing also raises concerns about the validity and robustness of the study’s findings.

Additionally, the authors conclude that latency reversal is enhanced when the SEC inhibitor KL-2 and LRAs such as JQ1 and AZD5582 are combined. However, only TAR and longLTR transcripts were measured, which does not provide a comprehensive assessment of the entire viral transcriptional profile. Lastly, there were no investigations into the latent reservoir through measurements of the viral DNA. The shock and kill strategy includes the clearance of the latently infected cells post-reactivation, therefore investigations into the effect of SEC inhibition may have benefitted from looking at the changes in the latent reservoir.

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