Screening for gene expression fluctuations reveals latency-promoting agents of HIV

Abstract

Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or “noise”) that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration–approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.

Synergistic Chromatin-Modifying Treatments Reactivate Latent HIV and Decrease Migration of Multiple Host-Cell Types

“…All flow cytometry data were analyzed using FCS Express 6 (Figure S2). A gate for JLat or TLat reactivation in the side scatter vs. fluorescein isothiocyanate (FITC) scatter plot was created using the untreated measurement similar to previous reports [6,20].…”

“…Excess over Bliss (EoB) scores were calculated for each treatment to determine whether a drug's interaction with TNF is additive or non-additive [6,9,22]. The EoB is defined as the difference between the observed combined effect and the expected combined effect:…”

“…Previous investigations have shown that the HIV LTR promoter displays episodic transcriptional bursting and that its decision making between active replication and latency is governed by stochastic fluctuations in viral gene expression [11][12][13][14] (Figure 1). In addition it has been shown that noise modulating compounds, including chromatin modifiers, are capable of synergizing with activators to generate more activation [6,9]. We simulated the two-state model of transcriptional bursting consisting of a promoter that can switch between a transcribing and non-transcribing state (Figure 1B) [14,25,26].…”

“…Successful application of LRA cocktails would enable the removal of ART after eradication of the latent reservoir. Alternatively, the "Block and Lock" treatment strategy aims to force integrated provirus into a deep latent state where it can no longer reactivate spontaneously [5][6][7], and offers a functional cure where antiretroviral therapy can be removed from infected individuals [5,8]. Both therapeutic strategies are being actively pursued and are currently an open paradigm in researching a cure for HIV.…”

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy

“…Reactivation of latent HIV in JLat 9.2 cells (Jordan et al, 2003) was previously studied using flow cytometry (Bohn-Wippert et al, 2017;Dar et al, 2014;Lu et al, 2021) and fluorescence microscopy (Bohn-Wippert et al, 2018), both using fluorescent probes expressed along with the viral genome. In a previous study (Bohn-Wippert et al, 2018), it was reported that reactivation occurred in cells with larger diameter.…”