DOI: 10.5061/dryad.qfttdz0t0
title: Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal

description: 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.
description: # Integrator complex subunit 12 knockout overcomes a transcriptional block
 to HIV latency reversal Dataset DOI:
 [10.5061/dryad.qfttdz0t0](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)