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

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.