Methylation of Epstein–Barr virus Rta promoter in EBV primary infection, reactivation and lymphoproliferation

Abstract

During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc.

The roles of DNA methylation on the promotor of the Epstein–Barr virus (EBV) gene and the genome in patients with EBV-associated diseases

“…Some studies have reported that the methylation status of the EBV promoter is related to the activation of the latent promoter. The EBV genome is unmethylated but becomes heavily methylated during the latent stage of the virus cycle in infected cells (Germi et al 2016 ). How EBV promoters obtain methylated cytosines is not yet clear; once established, this methylation pattern is maintained in proliferating and latently infected cells (Kalla et al 2012 ).…”

Comparison of immune responses in children with infectious mononucleosis caused by Epstein–Barr virus at different infection stages

“…On the other hand, since EBV infection is very common, the presence of NA IgG and EBV DNA may be reactivation infection, secondary infection or superinfection, which could not be distinguished in this study. According to previous studies, 17,31,32 we regarded the presence of NA IgG and EBV DNA as reactivation infection.…”

“…According to previous studies, 17,31,32 we regarded the presence of NA IgG and EBV DNA as reactivation infection.…”

Virological Markers in Epstein–Barr Virus-Associated Diseases

“…Currently, no straightforward technique is available to differentiate the intracellular episomal genome (found in latent infections and lymphoproliferations) from the viral linear genome (present in productive infections). Several studies suggest that the evaluation of EBV genome methylation may be an interesting tool, as low methylation levels point to a productive replication process, whereas high methylation levels indicate a latent/proliferative process [ 123 , 127 , 128 ]. This will be detailed in the final section below.…”

“…EBV DNA methylation patterns varied according to the EBV-associated disease under consideration, which could differ between healthy individuals and patients with NPC [ 168 ]. A number of studies analyzing the whole virus methylome or a single gene methylation, showed that EBV methylation was lower in non-malignant tissue biopsies than in malignant tumor biopsies [ 123 , 128 , 169 , 170 ]. Lam et al developed a combined approach of plasma EBV DNA analysis, including the plasma EBV DNA methylome, and the use of the fractional concentration and size of plasma EBV DNA for NPC diagnosis.…”