Research Article: Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies the proteasome as a key contributor to HIV-1 latency

Date Published: January 15, 2019

Publisher: Public Library of Science

Author(s): Zichong Li, Jun Wu, Leonard Chavez, Rebecca Hoh, Steven G. Deeks, Satish K. Pillai, Qiang Zhou, Ronald C. Desrosiers.

http://doi.org/10.1371/journal.ppat.1007498

Abstract

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.

Partial Text

Transcriptional silence of integrated HIV-1 proviruses in a minority of infected CD4+ T cells is a key signature of the latent viral reservoirs that necessitate a lifelong antiretroviral therapy (ART) to maintain their silence [1,2]. Strategies to expose the latently infected cells for immune recognition and clearance in individuals on ART rely on latency reversing agents (LRAs) to reactivate proviral transcription [3,4,5]. To date, multiple clinical trials have tested a variety of LRAs that are dominated by histone deacetylase (HDAC) inhibitors and NF-κB agonists [6]. However, only modest increases in viral transcription with little to no reservoir reduction are induced by these drugs [7].

In this study, we have developed a CRISPRi-based screen to reiteratively enrich loss-of-function genotypes that promote HIV-1 transcription in latently infected CD4+ T cells. The identified hits include the not-so-surprising factors that suppress the NF-κB pathway (NFKBIA, CYLD) or interact with the HDAC complex (GON4L), as well as three unexpected proteasomal subunits. Our subsequent experiments employing RNAi to target these three and also two other core subunits of the proteasome and testing various proteasome inhibitors in two different cell line-based latency models as well as primary CD4+ T cells from HIV-infected individuals on suppressive ART all support the notion that targeting the proteasome is an effective strategy to expose latent HIV-1.

 

Source:

http://doi.org/10.1371/journal.ppat.1007498

 

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