Inhibitors of the Histone Methyltransferases EZH2/1 Induce a Potent Antiviral State and Suppress Infection by Diverse Viral Pathogens

Abstract

Epigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses.IMPORTANCE A significant proportion of the world's population is infected with herpes simplex virus. Primary infection and subsequent recurrent reactivation can result in diseases ranging from mild lesions to severe ocular or neurological damage. Herpesviruses are subject to epigenetic regulation that modulates viral gene expression, lytic replication, and latency-reactivation cycles. Thus, epigenetic pharmaceuticals have the potential to alter the course of infection and disease. Here, while the histone methyltransferases EZH2/1 are implicated in the suppression of herpesviruses, inhibitors of these repressors unexpectedly suppress viral infection in vitro and in vivo by induction of key components of cellular innate defense pathways. These inhibitors suppress infection by multiple viral pathogens, indicating their potential as broad-spectrum antivirals.

Keywords: Zika virus; antiviral; chromatin; epigenetics; herpesvirus; immune mechanisms; innate immunity.

FIG 1

Inhibitors targeting EZH2/1 suppress HSV-1 IE gene expression. (A) Schematic of the PRC2 complex containing the EZH2 or EZH1 histone methyltransferase. GSK126, GSK343, and UNC1999 inhibit the catalytic SET domain, while astemizole interferes with the interaction of EED and EZH2/1. (B to E) mRNA levels of viral (ICP4, ICP22, ICP27) and control cellular genes (SP1, TATA box binding protein [TBP]) in HFF cells treated with the vehicle or the concentrations of EZH2/1 inhibitors indicated and infected with HSV-1 (MOI, 2.0) for 1.5 h. Data are means ± SEM of at least two independent experiments. (F) Western blot assay of viral IE (ICP4, ICP27) and cellular (actin) proteins from HFF cells treated with GSK126, GSK343, UNC1999, astemizole, or the vehicle and infected with HSV-1 (MOI, 2.0) for 2 h. (G, H) HFF cells were infected with HSV-1 (MOI, 0.01) for 8 h, and then the vehicle, ACV, GSK126, GSK343, UNC1999, or astemizole was added for 12 h. (G) Cells were stained with anti-UL29 (green) and phalloidin-647 (F-actin, red). (H) Viral yields. Data are means ± SEM of four independent experiments. ***, P < 0.001 (ANOVA and Dunnett’s post hoc test).

FIG 2

EZH2/1 inhibitors reduce the number of transcriptionally active viral genomes. (A to C) Viral transcriptional foci in HFF cells treated with the vehicle, GSK126, or UNC1999 and infected with HSV-1 (MOI, 5.0) for 1.5 h. Cells were stained with anti-ICP4 (viral transcriptional foci, red) and 4',6-diamidino-2-phenylindole (DAPI, gray). (D) The number of transcriptional foci per cell. Data are means ± SEM of ≥110 cells/group. ***, P < 0.001 (ANOVA and Dunnett’s post hoc test). (E) ChIP assays showing HCF-1, RNAPII, and histone H3K4me3 levels associated with the viral IE (ICP0) and control cellular (GAPDH) promoters in HFF cells treated with the vehicle or GSK126 for 5 h and infected with HSV-1 (MOI, 2.0) for 1.5 h. Data are means ± SEM of three independent experiments. **, P < 0.01; ***, P < 0.001 (unpaired two-tailed t test).

FIG 3

Inhibition of EZH2/1 leads to induction of innate immunity. (A, B) HFF cells were treated with the vehicle, GSK126, or UNC1999 for 5 h. (A) Cells were stained with anti-PML antibody (red) and DAPI (gray). (B) The number of PML foci per cell. Data are means ± SEM of >391 cells counted per group. ***, P < 0.001 (ANOVA and Dunnett’s post hoc test). (C) IFN-α, IL-8, and control SP1 mRNA levels in cells treated with the vehicle or GSK126 for 5 h. Data are means ± SEM of five independent experiments. (D) Differential expression (≥1.5-fold) of human IFNs and IFN receptors in HFF cells treated with the vehicle or GSK126 for 4 h, as determined with a Profiler PCR array (n = 3). (E to G) HFF cells were treated with the vehicle, GSK126, or GSK343 for 4 h, and differential expression was assessed by microarray analyses (n = 3). (E) The number of ISGs (Interferome database) that are differentially regulated (≥2-fold) by GSK126 or GSK343. Genes differentially expressed (≥2-fold) in both GSK126- and GSK343-treated cells are shown. The number of ISGs that are differentially regulated (≥2-fold) by GSK126 and GSK343 is shown. (F) Supergroups of related overrepresented IPA pathways in GSK126-treated cells. (G) STRING network of genes induced in qPCR arrays (≥1.5-fold) and microarrays (≥2-fold). Confidence levels of inferred functional associations between protein nodes (circles) are indicated by the thickness of the connecting lines (edges). (H, I) HFF cells were treated with the vehicle or GSK126 for 1, 2, or 5 h, and differential expression was assessed by RNA-Seq (n = 3). (H) The number of ISGs (Interferome database) that are differentially regulated (≥2-fold) by GSK126. (I) STRING network of genes induced (≥2-fold) by GSK126 at 2 h.

FIG 4

EZH2/1 inhibitors suppress hCMV, Ad5, and ZIKV infections. (A to E) HFF cells were treated with the concentrations of GSK126 indicated for 5 h and infected with ZIKV for 40 h (A) Cells were stained with pan-Flavivirus MAb E60 (blue). (B, C) FFU counts and sizes. (D) The percentage of cells infected at days 1 and 2 and treated with the concentrations of GSK126 indicated. (E) Cells were treated either preadsorption (pre) or postadsorption (post), and the percentage of cells infected was determined at days 1 and 2. The data in panels B to E are means ± SEM of at least two independent experiments.

FIG 5

Inhibitors of EZH2/1 suppress primary HSV-1 infection and enhance immune cell recruitment in vivo. (A, B) Mice infected with HSV-1 (2 × 10⁵ PFU/eye) were treated topically with the vehicle, ACV, GSK126, or UNC1999 for the times indicated. (A) HSV-1 yields and DNA loads from eyes at 7 dpi (≥12 eyes/group). (B) Viral yields from eyes on the postinfection days indicated (22 eyes/group). (C) Viral DNA yields from nasal tissues of mice infected intranasally with HSV-1 (5 × 10⁵PFU) and treated i.p. with the vehicle, ACV, or GSK126 for 7 days (13 nares/group). Panels A to C: *, P < 0.05; **, P < 0.01 (ANOVA and Dunnett’s post hoc test). (D) Mice were infected with HSV-1 (2 × 10⁵ PFU/eye) and treated topically with the vehicle, ACV, or GSK126 for 7 days. Eye sections were costained with anti-HSV-1 antibody (red), anti-Ly6G (neutrophil) antibody (green), and DAPI (blue).

FIG 6

EZH2/1 inhibitors suppress HSV-1 during reactivation from latency and stimulate immune signaling pathways in a mouse ganglion explant model. Trigeminal ganglia from latently infected mice were explanted in the presence of the vehicle, ACV, GSK126, GSK343, or astemizole for 48 h. (A) Viral yields per ganglion. Data means ± SEM of ≥19 ganglia/group. ***, P < 0.001 (Wilcoxon matched-pair signed-rank test). (B) Ganglion sections were costained with anti-UL29 antibody (red) and DAPI (blue) and scored for individual UL29⁺ neurons (single) (C) and clusters of UL29⁺ cells (D). The data in panels C and D are means ± SEM of 10 ganglia/group. ***, P < 0.001; ns, not significant (ANOVA and Dunnett’s post hoc test). (E to F) RNA-Seq analyses of mock-infected and latently infected ganglia explanted in the presence of the vehicle or GSK126 for 12 h (three pools of five ganglia per group). (E) The number of ISGs (Interferome database) differentially regulated (≥2-fold) by GSK126. (F) STRING network of genes induced (≥2-fold) in HSV-infected ganglia by GSK126. The confidence levels of inferred functional associations between protein nodes (circles) are indicated by the thickness of the connecting lines (edges).