Enhancing HIV-1 latency reversal through regulating the elongating RNA Pol II pause-release by a small-molecule disruptor of PAF1C

Abstract

The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus–1 (HIV-1) latency. Using a molecular docking–based compound screen in silico and global sequencing–based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock–down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal

“…While KL-2 was sufficient to boost viral replication in activated, primary CD4+ T cells, in and of itself it displayed minimal reactivation potential in both cell line models of latency and in PBMCs from PLWH on suppressive ART. This finding is similar to our recent report of a novel inhibitor of the PAF1 complex (iPAF1C) that had minimal activity on its own, but greatly enhanced the reactivation potential of other LRAs (16). Both cases highlight the multifaceted nature of the blocks to viral gene expression that underlie the latent state as well as the limitations to single agent drug screening to identify promising, next-generation LRAs.…”

“…After transcriptional initiation, RNA Pol II synthesizes 20-60 nucleotides before stalling through a well-conserved process known as promoter-proximal pausing (15). Pausing is enforced by several negative elongation factors, including negative elongation factor (NELF), DRB Sensitivity Inducing Factor (DSIF), and the RNA Polymerase II Associated Factor 1 (PAF1) complex (16)(17)(18). Pause release is regulated by positive transcription elongation factor-b (P-TEFb), a heterodimeric protein complex composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CCNT1) (19)(20)(21).…”

Dissecting the dynamics of coordinated active transcriptional repression in a multicellular organism

“…Among them, RNA Polymerase IIassociated factor 1 (Paf1, also known as antimeros in Drosophila), plays a critical role in regulating pausing along with other functions during elongation 36 . Reducing Paf1 levels, through classical knockdown, acute degradation, or a small molecule inhibitor resulted in augmented pause release genome-wide [36][37][38][39] . Based on these studies, we reasoned that a depletion of Paf1 in Drosophila embryos would diminish pausing and favor pause release, thus possibly affecting transcriptional repression.…”

The Archer and the Prey: The Duality of PAF1C in Antiviral Immunity

“…Being able to reconfigure this balance to benefit the virus or host could have powerful implications, and the contemporary identification of a small molecule inhibitor, referred to as iPAF1C, makes this a current reality. iPAF1C targets a key binding interface between CTR9 and PAF1 subunits, and the drug disrupted PAF1C chromatin localization and induced RNAPII release [ 70 ]. Moreover, the drug’s effect mimicked that of PAF1 depletion across the transcriptomic landscape.…”

“…This approach forces viral reactivation with latency reversal agents (LRAs) to purportedly clear reactivated cells via cytopathic effects or the host immune response [ 71 , 72 ]. iPAF1C enhanced the activity of existing LRAs in CD4+ T cells because the subsequent release of RNAPII advanced the transcriptional elongation of the provirus [ 70 ]. Importantly, iPAF1C alone could reverse latency in human peripheral blood mononuclear cells (PBMCs) derived from persons undergoing ART.…”