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. 2006 Nov;80(21):10700-11.
doi: 10.1128/JVI.01204-06. Epub 2006 Aug 18.

Methylation status of the Epstein-Barr virus (EBV) BamHI W latent cycle promoter and promoter activity: analysis with novel EBV-positive Burkitt and lymphoblastoid cell lines

Isabel A Hutchings  1 , Rosemary J Tierney, Gemma L Kelly, Julianna Stylianou, Alan B Rickinson, Andrew I Bell
Affiliations

Methylation status of the Epstein-Barr virus (EBV) BamHI W latent cycle promoter and promoter activity: analysis with novel EBV-positive Burkitt and lymphoblastoid cell lines

Isabel A Hutchings et al. J Virol. 2006 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
(A) Diagrammatic representation of the Cp- and Wp-initiated EBNA transcripts expressed in latency III LCLs. (B) Analysis of Wp and Cp activity in recently infected B cells. The graphs show the results of quantitative RT-PCR assays specific for Wp-initiated and Cp-initiated transcripts, expressed relative to an appropriate reference cell line. Results from one representative experiment for B cells harvested at 12 h and 2, 5, 8, 11, 14, 21, and 75 days postinfection are shown. Error bars indicate standard deviations for results of duplicate assays. (C) Diagram illustrating the main regulatory elements of Wp and the relative positions of the CpG dinucleotides analyzed. Shown are previously identified upstream activating sequences UAS2 and UAS1 which include binding sites for YY1, BSAP, RFX, and CREB, together with a recently identified second YY1 site between −270 and −276 relative to the transcription start site. Also marked are 20 CpG dinucleotides (black lollipop-shaped symbols, a to t) which represent potential methylated cytosines (B95.8 coordinates 13956, 13976, 14015, 14077, 14085, 14101, 14103, 14105, 14115, 14143, 14161, 14259, 14261, 14288, 14290, 14296, 14381, 14391, 14445, and 14462). CpG sites j, k, and n to p (boxed) have been shown previously to abrogate factor binding when methylated. (D) Results of bisulfite sequencing analysis of Wp in B cells 8 to 28 days postinfection. The Wp regulatory region was PCR amplified, cloned, and sequenced. Bisulfite-treated DNA was amplified with primers specific for Wp, and several PCR clones were sequenced for each sample. Individual CpG dinucleotides are identified as either methylated (+, shaded) or unmethylated (−).
FIG. 2.
FIG. 2.
Analysis of Wp and Cp transcription in established LCLs. Histograms show the results of quantitative RT-PCR assays to measure Wp-initiated and Cp-initiated transcripts. Error bars indicate standard deviations for duplicate assays. Also shown are mean EBV genome loads for the corresponding acyclovir-treated cell lines determined by quantitative DNA PCR using a primer-probe combination specific for the EBV BALF5 gene.
FIG. 3.
FIG. 3.
Bisulfite sequencing analysis of Wp in Wp-only LCLs and standard Cp/Wp-using LCLs. Bisulfite-treated DNA was amplified with primers specific for Wp, and several PCR clones were sequenced for each sample. Individual CpG dinucleotides are identified as either methylated (+, shaded) or unmethylated (−). EBH41.2 and IM100.1 share a sequence polymorphism (x) such that CpG site a is not present in Wp. Shown at the top is a diagram illustrating the main regulatory elements of Wp and the relative positions of the CpG dinucleotides analyzed.
FIG. 4.
FIG. 4.
(A) Design of the MSP assay used to analyze Wp methylation status. Shown is a diagram of the main regulatory elements of Wp, together with positions of the primers used in MSP analysis. (B) Results of MSP analysis of Wp methylation status in established LCLs. Bisulfite-treated DNA was amplified using primers specific for methylated (M) and unmethylated (U) Wp sequences, and the results were visualized on ethidium bromide-stained agarose gels. DNA from B cells 1 day postinfection served as a positive control for unmethylated sequences, while DNA from Akata-BL served as a positive control for methylated sequences.
FIG. 5.
FIG. 5.
(A) Schematic diagram of the recombinant B95.8 genome carrying either 11 BamHI W repeats (11W EBV) or 2 BamHI W repeats (2W EBV). The recombinant EBV genome also contains genes encoding hygromycin resistance (HygR) and green fluorescent protein (GFP). Also marked are the origin of plasmid replication (oriP) and terminal repeats (TR). (B) Analysis of Wp, Cp, and EBNA1 transcription in 2W and 11W LCLs. The histograms show the results of quantitative RT-PCR assays used to measure Wp-initiated, Cp-initiated, and BamHI Y3-U-K-spliced EBNA1 transcripts. Error bars indicate standard deviations of duplicate assays. (C) Western blot analysis for expression of EBV latent antigens EBNA1, EBNA-LP, EBNA2, and LMP1 in 2W and 11W LCLs.
FIG. 6.
FIG. 6.
(A) Bisulfite sequencing analysis of Wp in 2W and 11W LCLs. Data are presented as described in the Fig. 3 legend. (B) Results of MSP analysis of Wp methylation status in 2W, 4W, 6W, 8W, and 11W LCLs. Data are presented as described in the Fig. 4 legend.
FIG. 7.
FIG. 7.
(A) Diagrammatic representation of three programs of EBV latent gene expression found in different Awia-BL clones. Conventional latency I clones express EBNA1 alone from the BamHI Q promoter Qp. Atypical Wp-restricted clones carrying only the EBNA2-deleted form of the genome express EBNA1, -3A, -3B, -3C, and -LP from the BamHI W promoter Wp. Novel EBNA2+ LMP1− clones express all six EBNAs from an unidentified promoter in the absence of the LMPs. (B) Analysis of EBV latent gene expression in BL lines and Awia-BL clones. The histograms show the results of quantitative RT-PCR assays used to measure BamHI Q-U-K-spliced EBNA1, Wp-initiated, Cp-initiated, and EBNA2 transcripts. Error bars indicate standard deviations of results of duplicate assays. Also shown are mean EBV genome loads for the corresponding acyclovir-treated cell lines determined by quantitative DNA PCR using a primer-probe combination specific for the EBV BALF5 gene. Included as controls were the standard Cp/Wp-using LCLs IM100.1 and CD+Oku.
FIG. 8.
FIG. 8.
(A) Bisulfite sequencing analysis of Wp in latency I Akata-BL and Wp-restricted clones of Ava-BL, Oku-BL, and Sal-BL. Data are presented as described in the Fig. 3 legend. (B) Results of MSP analysis of Wp methylation status in latency I Akata-BL and Wp-restricted clones of Ava-BL, Oku-BL, and Sal-BL. Data are presented as described in the Fig. 4 legend.
FIG. 9.
FIG. 9.
(A) Bisulfite sequencing analysis of Wp in latency I, Wp-restricted, and EBNA2+ LMP1− Awia-BL clones. Data are presented as described in the Fig. 3 legend. (B) Results of MSP analysis of Wp methylation status in latency I Akata-BL and Wp-restricted clones of Ava-BL, Oku-BL, and Sal-BL. Data are presented as described in the Fig. 4 legend.
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