Epigenetic modification of the Epstein-Barr virus BZLF1 promoter regulates viral reactivation from latency

Abstract

The Epstein-Barr virus (EBV) is an oncogenic human gamma-herpesvirus that predominantly establishes latent infection in B lymphocytes. Viral genomes exist as extrachromosomal episomes with a nucleosomal structure. Maintenance of virus latency or execution of reactivation is controlled by the expression of BZLF1, a viral immediate-early gene product, tightly controlled at the transcriptional level. In this article, we review how BZLF1 transcription is controlled, in other words how virus reactivation is regulated, especially in terms of epigenetics. We recently found that histone H3 lysine 27 trimethylation (H3K27me3) and H4K20me3 markers are crucial for suppression of BZLF1 in latent Raji cells. In addition, H3K9me2/3, heterochromatin protein 1, and H2A ubiquitination are associated with latency, whereas positive markers, such as higher histone acetylation and H3K4me3, are concomitant with reactivation. Since lytic replication eventually causes cell cycle arrest and cell death, development of oncolytic therapy for EBV-positive cancers is conceivable using epigenetic disruptors. In addition, we note the difficulties in analyzing roles of epigenetics in EBV, including issues like cell type dependence and virus copy numbers.

Keywords: BZLF1 gene; Epstein–Barr virus; epigenetics; latency; reactivation.

FIGURE 1

Effects of an HDAC inhibitorTSA on BZLF1 expression differ with the type of EBV-positive cell. Akata, Raji, B95-8, and GTC-4 cells were treated with either vehicle (Cont) or 300 nMTSA. Anti-IgG (for Akata) or TPA/A23187/butyrate (for B95-8, Raji, and GTC-4) served as positive controls, as these substances induce BZLF1. After 24 h, RNAs were collected and real-time RT-PCR was carried out to measure the levels of BZLF1 mRNA, the results being shown as bars after normalization to levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. TSA alone treatment induced BZLF1 expression in Akata, but did not appreciably enhance in other cells.

FIGURE 2

Histone modification pattern of EBV BZLF1 promoter upon lytic reactivation. Raji cells were treated with control vehicle (blue) or TPA/A23187/butyrate (T/A/B, red) at 20 ng/ml, 1 μM and 5 mM, respectively. ChIP experiments were performed using antibodies indicated below, followed by real-time PCR. Active marks (H3Ac, H3K4me3) were elevated while levels of suppressive marks (H3K27me3, H4K20me3, and H3K9me2/3) remained unchanged.

FIGURE 3

Effects of pharmacological inhibitors on BZLF1 expression in Raji cells. Raji cells were treated with vehicle (Cont), 10 μM DZNep, 300 nMTSA, 1 μM 5-Aza alone or in combinations as indicated. As a positive control (TPA/A/Bu), Raji cells were treated with TPA/A23187/butyrate at 20 ng/ml, 1 μM and 5 mM, respectively. For DZNep or 5-aza-2′-deoxycytidine (5-Aza) treatment, cells were exposed to the reagent daily for 3 days. Treatment with other chemicals was for 24 h. Real-time RT-PCR was carried out to measure the levels of BZLF1 mRNA, which were then normalized to GAPDH mRNA levels. The light-blue arrows indicate that DZNep alone did not efficiently induce BZLF1, but could markedly enhance the expression if treated in combination with TSA.

FIGURE 4

Summary of epigenetic histone modifications in the BZLF1 promoter of Raji cells. Repressive histone H3K9, H3K27, H4K20 methylations (marked with blue circles) are present in latency, and are not appreciably decreased even after induction. High levels of active markers, such as H3K4 methylation and histone acetylation (green circles), are notably associated with lytic induction.