Epigenetic regulation of EBV genome is a fundamental regulatory mechanism determining different types of EBV infections in its associated tumors [5-8]. Several latent or lytic genes, including EBV nuclear antigens (EBNA-2, 3A, 3B, 3 C), latent membrane protein 1 (LMP1), IE antigens (Zta, Rta) and lytic cycle viral kinases, have been identified tightly controlled by the CpG methylation of various EBV promoters [1,5,9,10], such as W promoter (Wp), C promoter (Cp), Q promoter (Qp), F promoter (Fp), LMP1 promoters (ED-L1 and ED-L1E promoters), Z promoter (Zp) and R promoter (Rp). The precise epigenetic regulation ensures the production of viral progeny without releasing viral antigens detectable by host immune system. Meanwhile, reactivation of viral genes from latency by demethylation agents could serve as a therapeutic strategy for EBV-associated tumors [1,10-12].

We also demonstrated that demethylation of Zp and Rp by treatment with 5-aza-dC alone or combined with TSA resulted in the re-expression of BZLF1 and BRLF1 and activation of EBV lytic cycle. It has been identified that DNA synthesis inhibitors have no effect on DNA methylation by using four different inhibitors of DNA replication [29]. Although DNA synthesis inhibitors will delay some of the cytosine methylation, all delayed DNA methylation will be finally completed prior to the subsequent S phase. Thus, in our study, DNA methylation inhibitors are mainly responsible for Zp and Rp demethylation and initiation of lytic cascade, while other events indirectly leading to EBV reactivation also cannot be ruled out.

In EBV-positive cell lines except for B95-8, Wan and AG876, only very few % of cells or no cell undergoing spontaneous lytic infection without much lytic virion DNA, thus the methylation status of Zp and Rp detected represents the latent viral genome but not the virion genome, while in B95-8, Wan and AG876 cell lines with significant % of cells undergoing spontaneous lytic infection, the methylation status detected probably represents both latent and viral genomes (Figure 2C, D). Similarly, in EBV-positive tumors with rare cell undergoing spontaneous lytic infection, methylation status represents the latent genome [1]. In our study, lower level of methylation was only observed in B95-8 and Wan cell lines, but not in other EBV-positive cell lines and primary tumors, consistent with the high level of spontaneous EBV lytic replication only in B95-8 and Wan.

The existence of a small proportion of cells expressing viral lytic genes is essential for the success maintenance of EBV latency in host cells with a highly methylated viral genome [1,3]. As viral transactivator proteins, Zta is unique to initiate the entire EBV lytic cascade by transactivating a series of lytic gene promoters, but Rta appears to be more effective in epithelial cells [30,31]. Increased evidences have shown that Zta initiates EBV lytic infection mainly from a methylated viral genome, whereas Rta initiates lytic infection mainly from an unmethylated genome [15,16,32,33]. Rp methylation inhibits Rta expression, however it enhances the ability of Zta to activate Rp [16]. In line with reported studies, we found that either the Rp-ZRE2 (CpG site #13-14) or/and the ZRE3 (CpG site #15) were heavily methylated in virtually all EBV-positive BL, LCL and NPC cell lines and tumors, but less methylated in Wan and B95-8 cells with basal lytic activity. A CpG methylation-free zone (three CpG sites #12-14) in Zp, located in regulatory elements YY1 and E2-2, is possibly responsible for the initial activation of BZLF1. Thus, Zp and Rp are regulated by both CpG methylation and cellular transcription factors, indicating the complexity of the regulation of BZLF1 and BRLF1 in EBV-associated tumors [34].

DNA methylation plays a crucial role in allowing EBV to escape from the detection of host immune system. Conversely, pharmacologic reversal of viral gene methylation and activation of gene expression would resensitize host's immune surveillance or enhance its response to immunogenic viral antigens [10]. The efficacy of DNA methyltransferase inhibitors in hematologic diseases, including myelodysplastic syndromes (MDS) and acute myeloid leukemia, has been successfully evaluated by multiple clinical trials [35-37]. 5-azacytidine has been now approved as the first-line treatment of high-risk MDS [38,39]. Our group has firstly reported the achievement of successful demethylation of EBV viral antigen promoters including Cp, Wp, LMP1p, Zp and Rp, in NPC patients with azacitidine treatment [1,10-12]. When combined with other chemotherapy drugs and histone deacetylase inhibitors (HDACi), DNMT inhibitors should have even brighter perspective in the therapeutics of EBV-associated tumors (Figure ​(Figure7)7) [40].