Methylation Status of theEpstein-Barr Virus (EBV) BamHI W Latent Cycle Promoter and Promoter Activity: Analysis with Novel EBV-Positive Burkitt and LymphoblastoidCell Lines

Abstract

The Epstein-Barr virus (EBV) latent cycle promoter Wp, present in each tandemly arrayed copy of the BamHI W region in the EBV genome, drives expression of the EB viral nuclear antigens (EBNAs) at the initiation of virus-induced B-cell transformation. Thereafter, an alternative EBNA promoter, Cp, becomes dominant, Wp activity declines dramatically, and bisulfite sequencing of EBV-transformed lymphoblastoid cell lines (LCLs) shows extensive Wp methylation. Despite this, Wp is never completely silenced in LCLs. Here, using a combination of bisulfite sequencing and methylation-specific PCR, we show that in standard LCLs transformed with wild-type EBV isolates, some Wp copies always remain unmethylated, and in LCLs transformed with a recombinant EBV carrying just two BamHI W copies, Wp is completely unmethylated. Furthermore, we have analyzed rare LCLs, recently established using wild-type EBV isolates, and rare Burkitt lymphoma (BL) cell clones, recently established from tumors carrying EBNA2-deleted EBV genomes, which express EBNAs exclusively from Wp-initiated transcripts. Here, in sharp contrast to standard LCL and BL lines, all resident copies of Wp appear to be predominantly hypomethylated. Thus, studies of B cells with atypical patterns of Wp usage emphasize the strong correlation between the presence of unmethylated Wp sequences and promoter activity. Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus implicated in the pathogenesis of several human malignancies, efficiently transforms resting B cells in vitro into permanently proliferating lymphoblastoid cell lines (LCLs). Such LCLs all display a latency III form of infection characterized by the constitutive expression of six EBV-encoded nuclear antigens, EBNA1, -2, -3A, -3B, -3C, and -LP, and three latent membrane proteins, LMP1, -2A, and -2B (38). While the LMP genes are transcribed from their own individual promoters, all six EBNA mRNAs are generated by the splicing of long primary transcripts which initiate from one of two alternative promoters, Wp or Cp (9,10,41,47). Wp, present in each BamHI W repeat of the EBV genome, is selectively activated immediately postinfection (3, 56). While these Wp-initiated transcripts can potentially encode all six EBNAs, at these early time points there appears to be a preferential expression of EBNA2 and EBNA-LP; subsequently, these two antigens activate the alternative EBNA promoter, Cp (39, 48, 54), leading to the broadening of virus antigen expression to all six EBNAs, and upregulate the expression of the LMP promoters (1,17,53,62). The early stages of B-cell transformation are, therefore, characterized by a marked switch in EBNA promoter usage, with Cp becoming dominant over Wp, leading to the outgrowth of 55,56).While there has been some progress in identifying the cellular factors important for the initial activation of Wp in resting B cells (8,28,50), how Wp is subsequently repressed remains poorly understood. This is an important question, however, since the flexibility of latent promoter usage is central to ...

Chromatin Profiling of Epstein-Barr Virus Latency Control Region

“…DNA methylation of key regulatory elements within the Cp correlates with transcription repression during type I latency (47). However, the kinetics of DNA methylation revealed that this event occurs subsequent to transcriptional repression, suggesting that DNA methylation maintains but does not initiate the switch from type III to type I gene expression (27).…”

“…Latency type switching provides the virus with a strategy to stimulate B-cell proliferation and subsequently avoid host immune detection and elimination of infected cells (62). Latency type selection is known to be influenced by cell-specific transcription factors as well as by epigenetic events, including DNA methylation, histone modifications, and chromatin organization (3,12,17,27,43).…”

Contributions of CTCF and DNA Methyltransferases DNMT1 and DNMT3B to Epstein-Barr Virus Restricted Latency

“…Interestingly, following infection of GC B cells, DNMT3A could be detected within chromatin associated with Wp (but not Cp) (28), the EBNA promoter used prior to EBNA2 transactivation of EBNA expression from Cp. Most importantly, EBV-mediated induction of DNMT3A and its direct association with Wp, a latency gene promoter that undergoes methylation relatively early in infection (16) and which must ultimately be silenced to establish and maintain restricted latency, support the notion that methylation of the EBV genome is a regulated process. It will be interesting to determine, therefore, whether DNMT3A plays a critical role in the establishment of restricted latency beyond methylation of Wp.…”

“…Subsequent investigations identified methylated CpG residues within latency gene promoters that either correlated with transcriptional inactivity, inhibited transcription, or prevented binding by key transcriptional activators (17,45,47,48,52,53,56,62) and that in some instances were found to be actually methylated within peripheral blood B cells isolated from healthy EBV-infected individuals (37,46). Collectively, these studies have provided strong evidence that DNA methylation is critical to establishment of restricted latency programs, though CpG methylation has been observed to lag behind transcriptional downregulation of at least one of the EBNA gene promoters (Wp) that is silent during restricted latency (16). Thus, methylation of the EBV genome may be more critical to maintenance of a transcriptionally silent state than to its initiation, though how it may be specifically regulated has yet to be determined.…”

“…Total cellular RNA was extracted using RNA-BEE (Tel-Test) according to the manufacturer's instructions and treated with RQ1-DNase (Promega) to remove residual DNA. cDNA was generated from 2 g total RNA in 20-l reaction mixtures with 200 U SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions, using either 0.1 pmol gene-specific primer (GSP) for EBV transcripts or 5 ng oligo(dT) [12][13][14][15][16][17][18] (Invitrogen) for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and DNMT3B mRNAs. Nucleotide sequences and descriptions of primers used for RT-PCR are provided in Table S1 in the supplemental material.…”