Bromodomain proteins regulate human cytomegalovirus latency and reactivation allowing epigenetic therapeutic intervention

Abstract

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.

Keywords: I-BET; bromodomain proteins; cytomegalovirus; epigenetics; latency.

Fig. 1.

HDAC and BET inhibitors induce high numbers of IE86-expressing cells in the THP-1 cell line model of HCMV latency. (A) Peripheral blood CD14+ monocytes from three HCMV seropositive epilepsy patients being treated with VPA and from eight controls were analyzed for HCMV copy number by ddPCR. (B and C) Monocytic THP-1 cells infected with HCMV TB40e IE86-eYFP for 3 d were treated with inhibitors of histone acetyltransferases (HATi, green), histone deacetylases (HDACi, red), bromodomain proteins (BRDi/I-BET, blue), histone demethylases (HDMi, orange), histone methyltransferases (HMT inhibitor, teal), as well as other epigenetic modifiers (Other, pink) across a range of concentrations (30 μM to 30 nM) for 48 h before the number of YFP-positive cells was analyzed by flow cytometry in comparison to DMSO (negative control, set to 0) and PMA (100 nM, gray dashed line) (mean + SEM, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). (Inset) An example image of cells counted (20× magnification).

Fig. 2.

BET inhibitors induce virus gene expression while restricting full reactivation from ex vivo HCMV latency models. (A–C) CD14+ monocytes isolated from apheresis cones were infected with HCMV TB40e IE86-eYFP for 5 d before treatment with HDACi (red bars), BRDi/I-BET (blue bars), or a dual inhibitor (purple bars) across a range of concentrations (30 μM to 30 nM) for 72 h (A). The number of YFP-positive cells per well was then counted (B) before monocytes were overlaid with HFFFs and the resulting plaques were counted after 7 d (C) (mean + SEM, n = 3). (D) CD14+ monocytes isolated from apheresis cones were treated with inhibitors for 72 h before being stained with SYTOX (dead cells, red stain) and Hoechst stain (nucleus, blue stain) and counted (mean + SEM, n = 3) († = total cell death). (Insets) Example images of each condition counted (20× magnification). (*P < 0.05, **P < 0.01, ***P < 0.001.)

Fig. 3.

BET inhibitors dysregulate HCMV transcription from latency and restrict viral DNA replication. (A–E) CD14+ monocytes isolated from apheresis cones infected with HCMV TB40e wild type for 5 d were treated with DMSO (black lines), HDACi (CHR-4487 30 nM, red lines), I-BET (GSK726 30 nM, blue lines) and PMA (20 nM, green lines). RNA, DNA, and protein were isolated from cells every 24 h for 4 d. RT-qPCR was then performed for virus (A) IE72, (B) early UL44, and (C) late UL83/pp65 transcripts relative to host GAPDH with (D) HCMV genome copy number determined by qPCR of UL44 promoter relative to host GAPDH promoter and (E) immunoblotting carried out for virus targets relative to β-actin (images representative of n = 3). *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM, n = 3. (F–H) RNA-seq of flow cytometry isolated YFP-positive HCMV-TB40e-IE86-eYFP–infected CD14+ monocytes after 72 h treatment with compounds (F). Data are presented as a heat map of means of HCMV transcripts identified from biological duplicates, with data ranked by I-BET (GSK726) fold-change relative to DMSO control. HCMV gene groups indicated by associated asterisk color (red, structural protein; green, long noncoding RNA; orange, immunomodulatory protein; blue, DNA replication protein). Venn diagrams show HCMV transcript number from RNA-seq data either (G) up-regulated (>1.5-fold) or (H) down-regulated (>50%) relative to DMSO control.

Fig. 4.

BRD4 inhibition modulates SEC:P-TEFb association with HCMV promoters. (A–H) Monocytic THP-1 cells infected with HCMV TB40e IE86-eYFP for 3 d were treated with DMSO (black bars), HDACi (CHR-4487 30 nM, red bars), I-BET (GSK726 30 nM, blue bars) or PMA (100 nM, green bars). After 48 h, ChIP qPCR analysis was employed to determine the enrichment of (A–D) MIEP and (E–H) UL44 promoter DNA using antibodies specific for (B and F) CDK9, (C and G) BRD4, and (D and H) RNAPII S2P relative to isotype controls (dashed line, set to 1) (mean + SEM, n = 3). (I) Treatment regime of cells in J–K. CD14+ monocytes isolated from apheresis cones infected with HCMV TB40e IE86-eYFP for 5 d were treated for 72 h with DMSO (black bars), HDACi (CHR-4487, red bars), or I-BET (GSK726, blue bars) (all 30 nM) with concurrent treatment of half of wells with either (J) CDK9 inhibitor (CDK9i), Flavopiridol (FLAV 40 nM), or (K) SECi KL-2 (10 μM) before the number of YFP-positive cells per well were then counted. (L–N) CD14+ monocytes isolated from apheresis cones infected with HCMV TB40e IE86-eYFP for 5 d were treated with DMSO (black bars) or BRD4-degrader dBET1 (blue bars) (3 to 0.03 μM) with immunoblot data confirming BRD4 degradation at 24 h (L) (images representative of n = 3). After 72 h, the number of YFP-positive cells per well then counted (M) before monocytes were overlaid with HFFFs and resulting plaques counted after 7 d (N) (mean + SEM, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5.

BET inhibitors allow efficient killing of latent HCMV-infected cells and reduce peripheral blood virus carriage. (A–D) CD14+ monocytes isolated from seropositive peripheral blood were infected with HCMV TB40e UL32-GFP for 5 d before cocultures were established (no cells, full bars; CD14-depleted PBMCs, dotted bars; CD14/CD4/CD8-depleted PBMCs, hatched bars) and treated with either DMSO (black bars), HDACi (CHR-4487, red bars), or I-BET (GSK726, blue bars) (30 and 3 nM) for 72 h (A). The number of GFP-positive cells per well was then counted for seropositive donor (B) CMV301, (C) CMV302, and (D) CMV303 (mean + SEM, n = 3). (E and F) Experiments were repeated with additional coculture of CD14/CD3-depleted PBMCs (checkered bars), and GFP-positive cells were counted for (E) seropositive donor CMV301 and (F) seronegative donor CMV400 (mean + SEM, n = 4; *P < 0.05, **P < 0.01, ***P < 0.001). (G) Treatment regime of cells in H. CD14+ monocytes isolated from peripheral blood of five seropositive donors were plated for 3 h before cocultures were established using total remaining PBMCs and either DMSO (black symbol) or I-BET (GSK726, 30 nM; blue symbol). After 4 d, coculture was withdrawn and CD14+ monocytes were differentiated (GM-CSF/IL-1β 5-d, LPS 5-d) before cells were harvested, DNA isolated and (H) HCMV genome copy number determined by qPCR of glycoprotein B DNA relative to host GAPDH promoter (mean, n = 3). **P value = 0.0092 (Student’s t test).