Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal

Reviewer #1 (Public review):

Gray and colleagues describe the identification of Integrator complex subunit 12 (INTS12) as a contributor to HIV latency in two different cell lines and in cells isolated from the blood of people living with HIV. The authors employed a high-throughput CRISPR screening strategy to knock down genes and assess their relevance in maintaining HIV latency. They had used a similar approach in two previous studies, finding genes required for latency reactivation or genes preventing it and whose knockdown could enhance the latency-reactivating effect of the NFκB activator AZD5582. This work builds on the latter approach by testing the ability of gene knockdowns to complement the latency-reactivating effects of AZD5582 in combination with the BET inhibitor I-BET151. This drug combination was selected because it has been previously shown to display synergistic effects on latency reactivation.

The finding that INTS12 may play a role in HIV latency is novel, and the effect of its knockdown in inducing HIV transcription in primary cells, albeit in only a subset of donors, is intriguing. However, there are some data and clarifications that would be important to include to complement the information provided in the current version of the manuscript.

Reviewer #2 (Public review):

Summary:

Identifying an important role for the Integrator complex in repressing HIV transcription and suggesting that by targeting subunits of this complex specifically, INTS12, reversal of latency with and without latency reversal agents can be enhanced.

Strengths:

The strengths of the paper include the general strategy for screening targets that may activate HIV latency and the rigor of exploring the mechanism of INTS12 repression of HIV transcriptional elongation. I found the mechanism of INTS12 interesting and maybe even the most impactful part of the findings.

Weaknesses:

I have two minor comments:

There was an opportunity to examine a larger panel of latency reversal agents that reactivate by different mechanisms to determine whether INTS12 and transcriptional elongation are limiting for a broad spectrum of latency reversal agents.

I felt the authors could have extended their discussion of how exquisitely sensitive HIV transcription is to pausing and transcriptional elongation and the insights this provides about general HIV transcriptional regulation.

Reviewer #3 (Public review):

Summary:

Transcriptionally silent HIV-1 genomes integrated into the host`s genome represent the main obstacle to an HIV-1 cure. Therefore, agents aimed at promoting HIV transcription, the so-called latency reactivating agents (LRAs) might represent useful tools to render these hidden proviruses visible to the immune system. The authors successfully identified, through multiple techniques, INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency and hindering elongation of the HIV RNA transcripts. This factor synergizes with a previously identified combination of LRAs, one of which, AZD5582, has been validated in the macaque model for HIV persistence during therapy (https://pubmed.ncbi.nlm.nih.gov/37783968/). The other compound, I-BET151, is known to synergize with AZD5582, and is a inhibitor of BET, factors counteracting the elongation of RNA transcripts.

Strengths:

The findings were confirmed through multiple screens and multiple techniques. The authors successfully mapped the identified HIV silencing factor at the HIV promoter.

Weaknesses:

(1) Initial bias:
In the choice of the genes comprised in the library, the authors readdress their previous paper (Hsieh et al.) where it is stated: "To specifically investigate host epigenetic regulators involved in the maintenance of HIV-1 latency, we generated a custom human epigenome specific sgRNA CRISPR library (HuEpi). This library contains sgRNAs targeting epigenome factors such as histones, histone binders (e.g., histone readers and chaperones), histone modifiers (e.g., histone writers and erasers), and general chromatin associated factors (e.g., RNA and DNA modifiers) (Fig 1B and 1C)".

From these figure panels, it clearly appears that the genes chosen are all belonging to the indicated pathways. While I have nothing to object to on the pertinence to HIV latency of the pathways selected, the authors should spend some words on the criteria followed to select these pathways. Other pathways involving epigenetic modifications and containing genes not represented in the indicated pathways may have been left apart.

(2) Dereplication:
From Figure 1 it appears that INTS12 alone reactivates HIV -1 from latency alone without any drug intervention as shown by the MACGeCk score of DMSO-alone controls. If INTS12 knockdown alone shows antilatency effects, why, then were they unable to identify it in their previous article (Hsieh et al., 2023)? The authors should include some words on the comparison of the results using DMSO alone with those of the previous screen that they conducted.

(3) Translational potential:
In order to propose a protein as a drug target, it is necessary to adhere to the "primum non nocere" principle in medicine. It is therefore fundamental to show the effects of INTS12 knockdown on cell viability/proliferation (and, advisably, T-cell activation). These data are not reported in the manuscript in its current form, and the authors are strongly encouraged to provide them.

Finally, as many readers may not be very familiar with the general principles behind CRISPR Cas9 screening techniques, I suggest addressing them in this excellent review: https://pmc.ncbi.nlm.nih.gov/articles/PMC7479249/.

Author response:

We thank the reviewers for the positive and constructive feedback on our manuscript. We appreciate you highlighting the importance of our work in advancing our understanding of HIV latency and viral reactivation. The reviewers had mostly minor comments that we are in the process of addressing by completing additional experiments that are responsive to reviewer comments as well as some clarification of the text. These include:

(1) The impact of INTS12 knockout on cell viability.

We did not see an effect of the knockout of INTS12 on cell viability in the flow cytometry gating of live/dead cells, nor a gross difference in cell proliferation. However, we will test cell viability and proliferation more quantitatively and include this data in the revision.

(2) The effect of INTS12 knockout on additional LRAs.

There is published data that the Integrator complex inhibits HIV reactivation via additional LRAs that we will better highlight in the revision. In addition, we have data that we did not include in the original submission suggesting that INST12 knockout affects the degree of HIV reactivation with additional LRAs. We will confirm these results and include the data in the revision.

(3) Extend the discussion on how exquisitely sensitive HIV transcription is to pausing and transcriptional elongation and the insights this provides about general HIV transcriptional regulation.

Yes, we agree with this and will extend the discussion in this manner. We will also include additional data that we recently obtained that further emphasizes this point.

(4) Comparison to another CRISPR screen using the same library (Hsieh et al., PLOS Pathogens, 2023).

Indeed, INST12 was one of the hits in the previous paper (Hsieh et al., 2023) but was not specifically described or validated in that paper. We will point that out in the revision. Also, the Hsieh et al paper already described the library in more detail, but we will include additional text in the revision to emphasize that it casts a wide net on processes involved in transcriptional regulation.

(5) We made a mistake on the numbering of the supplemental figures which lead to some misunderstanding. We will correct this as well as add other suggestions of the reviewers for clarifications.