Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal
Data files
Apr 10, 2025 version files 2.50 MB
-
250406_DryadInformation_v2.xlsx
2.50 MB
-
README.md
1.25 KB
Abstract
The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). In this study, we performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12), as we see enrichment of this gene over all others. Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. We observed knockout of INTS12 improved latency reactivation at the transcriptional level in J-lat cells compared to the safe-harbor locus AAVS1 knockout. INTS12 knockout is also more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone as we see less host cell gene dysregulation compared to HIV induction. When we measured INTS12 binding by CUT&Tag we observe more INTS12 at the promoter of our control cells vs our INTS12 knockout cells, therefore its effect on HIV may be direct. Additionally, by CUT&Tag, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action, as we see more virus with INTS12 knockout than any of the LRAs/stimuli alone.
Dataset DOI: 10.5061/dryad.qfttdz0t0
Description of the data and file structure
This data was collected to assess the effects of INTS12 knockout on HIV latency reversal on its own and in the presence of AZD5582 & I-BET151.
Files and variables
File: 250323_DryadInformation.xlsx
Description:
There are 14 tabs total in this Excel:
Tab 1 is a read me with general information, Tabs 2-8 correspond to each main figure from the manuscript, example: Figure1, Tabs 9-14 correspond to each supplementary figure from the manuscript, example: Figure1_Supp1 means Figure 1 supplementary figure 1. Each tab contains all data pertaining to a specific figure (for instance Figure 1 will include Figure 1B and 1C information, as those have graphical data) in the closest format to how the data was graphed. For this reason, some values may be included in a separate column so that the exact graphs can be recreated from the manuscript.
Variables
- Knockout cells: INTS12 knockout or AAVS1 knockout (control)
- LRA treatments: PMA & Ionomycin (PMAi) , AZD5582 & I-BET151 (AZD & IBET), DMSO (control)