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

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.

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Review: 1.    In this study, the authors reported that knocking down SEC components did not dramatically impact HIV replication in primary CD4+ T cells, and the inhibition of P-TEFb/SEC interaction by KL-2 stimulated viral transcription. In HIV latency models in vitro, KL-2 acts synergistically with other LRAs to disrupt latent HIV, which in part is dependent on HIV Tat/TAR interaction. In PBMCs isolated from PWH on ART KL-2 enhanced HIV latency reversal in the presence of other LRAs. The authors claimed that the release of P-TEFb from cellular SECs may be an alternative approach for latency reactivation. Overall, the observations look interesting but mechanistic evidence is lacking with some obvious caveats, which could be improved.

Major points:

1. Overall, the concerns are that concentrations (3.125-6.5 µM) disrupting latent HIV cause undesired cellular toxicity. This greatly compromised the quality of the data overall.

2. In Fig 1D, can the authors also measure HIV transcription? If looking into the data more carefully, AFF1 and ENL KO significantly increased viral infection, although it was not dramatically.

3. In Fig 2, while using an interaction inhibitor with KL-2 is a good tool to study the role of SEC/P-TEFb in HIV transcription/latency, a very high dosage was used in order to achieve latency reversal. The issue was that at these dosages, KL-2 was very toxic to cells, making all the data suspicious. To avoid that, SEC KO could be pursued. 

4. In Fig 2G-H, usually, in the same HIV RNA, you should see more TAR HIV RNA fold change than the long LTR region since TAR RNA represents overall HIV transcription while gag represents elongation of HIV transcription. However, this was not observed here, which may reflect toxicity issues presumably caused by high levels of KL-2.

5. In Fig 3, again, a very high dosage of KL-2 (6.25 µM) was used, with known cellular toxicity (~50% cell death). With this, the data may not be trustful here. Again, the fold change of HIV RNA was similar between HIV TAR and long LTR. 

6. In Fig 4A, it might be good to KO Tat, but not apply nonspecific Tat inhibitors. TFIIH inhibitor Spironolactone is not specific to Tat. Data with U1 and AH2 cells could be explained as the interaction of TAR/Tat, but not insufficient Tat protein itself. In 4D, what will happen if KL-2 is added before Tat overexpression? 

7. The PWH data were interesting, but the mechanistic implications of AZD5582 plus KL-2 data could be complicated, unless tat gene expression or mutation was measured.