The Epstein-Barr Virus Major Latent Promoter Qp Is Constitutively Active, Hypomethylated, and Methylation Sensitive

Cell lines, 5-azacytidine treatment, and tumor samples.

Rael, Akata, and Chep are EBV-positive [EBV(+)] type I Burkitt’s lymphoma (BL) cell lines. Wan is a type I/II EBV(+) BL cell line. Wewak, Namalwa, Raji, and AG876 are type III EBV(+) BL cell lines. B95-8 is an EBV-immortalized LCL from marmoset B cells. IB4 is a B95-8 EBV-transformed human B-cell line (LCL). CA46 is an EBV-negative [EBV(−)] BL cell line. BJAB is an EBV(−) B-lymphoma cell line. Cell lines were maintained at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM glutamine, and 100 U of penicillin and streptomycin per ml. 5-Azacytidine (Sigma) was used at 1 μM (final concentration) to treat the Rael cell line (to generate Rael-AzaC cells) (44).

Endemic BL (eBL) specimens from Ghana, Africa, posttransplant lymphoproliferative disease (PTLD) specimens from the United States, nasopharyngeal carcinoma (NPC) specimens from Taiwan, and Hodgkin’s disease (HD) specimens from the United States have been previously described (45, 59). DNA and RNA of three cases of nasal lymphoma (NL) (two of NK cell origin [NK1 and -2] and one of T-cell origin [NTL]) have been studied previously (13).

Plasmid DNA.

Qp sequences (−172 to +32 relative to the Qp start site, coordinates 62250 to 62453 in the B95-8 genome) were amplified from B95-8 DNA by PCR and cloned into the pCR2.1 vector (Invitrogen) (Fig. 2A). The Qp fragment was then isolated and cloned into the HindIII and XbaI sites of a chloramphenicol acetyltransferase (CAT) reporter plasmid, pCAT-enhancer (Promega). The sequence and orientation of the insert in the recombinant plasmid pQpCAT were verified by dideoxy sequencing (U.S. Biochemical).

RT-PCR for EBNA1 transcripts.

Reverse transcription-PCR (RT-PCR) primers and internal probes for EBNA1 transcripts (FUK, QUK, and YUK) initiated from different promoters (Fp, Qp, Cp, and Wp) are listed in Table 1. Primers U′ and U2′ were used for the lytic Fp transcript (FUU′) (Fig. 1A). Total RNA was extracted from frozen tissues of EBV(+) tumors and fresh cell lines by using TriZol (Gibco BRL). Random hexamers and the GeneAmp RNA PCR kit (Perkin-Elmer) were used for reverse transcription and PCR. The PCR involved an initial denaturation at 95°C for 3 min, followed by 40 cycles (94°C for 30 s, 55°C for 1 min, and 72°C for 1 min), with a final extension at 72°C for 10 min. RNA from the EBV(−) cell line BJAB was used as negative control. The RT-PCR products were electrophoresed on a 1.8% agarose gel and transferred in 0.4 M NaOH onto a HyBond N(+) membrane. The membrane was hybridized by using a [γ-³²P]ATP-labeled internal oligonucleotide probe and the Rapid-Hyb buffer system (Amersham) at 52°C for 2 h. The membrane was then washed and autoradiographed. RT-PCR hybridization signal strength was graded as ++, +, or +/−, corresponding to strong signal after a 1-h exposure, strong signal after an overnight exposure, and weak signal after an overnight exposure, respectively (59).

Bisulfite genomic sequencing.

Genomic DNA was extracted from tumor tissues or cell pellets with DNAZol (Gibco BRL) or by the conventional proteinase K digestion method. Bisulfite DNA treatment was performed as described previously (17, 44). Briefly, the DNA was digested with EcoRI, denatured with NaOH, precipitated, and incubated with 3.1 M sodium bisulfite (Sigma) at 50°C for 16 h in darkness. After the reaction, DNA was desalted and purified by the Wizard DNA purification system (Promega). The DNA was then treated with 0.3 M NaOH and recovered. The bisulfite-treated DNA was PCR amplified with strand-specific primers (for the bisulfite-converted bottom strand): 5′-AACTAACCTAACTAAAAATAAAAC (corresponding to EBV coordinates 62179 to 62202) and 5′-AATGTAAGGATAGTATGTATTATT (corresponding to EBV coordinates 62481 to 62458). The PCR products were electrophoresed and purified by using Spin-X tubes (Costar). The PCR products were then cloned into the pCR2.1-TA cloning vector (Invitrogen). Four to six colonies were analyzed for each DNA sample. Plasmid DNA was extracted and sequenced.

Methylation cassette assay.

The methylation cassette assay was performed as described previously (42). Briefly, pQpCAT containing the entire Qp was methylated with CpG methylase SssI (New England Biolabs). Completeness of methylation was monitored by digestion of an aliquot with the methylation-sensitive restriction endonuclease HhaI. Methylated and unmethylated plasmid DNAs were then digested overnight with HindIII and XbaI and electrophoresed. Methylated and unmethylated Qp cassette and unmethylated background fragments were recovered by using a QiaexII gel extraction kit (Qiagen). In parallel, 2 μg of Qp cassette (methylated or unmethylated) and unmethylated background fragments were ligated in a 1:1 molar ratio at 16°C for 24 h with 10 U of T4 DNA ligase (Boehringer Mannheim) in a 20-μl volume. The ligation mixture was transfected by the DEAE-dextran method into the Rael and CA46 cell lines. After 2 days, cells were harvested for assay by thin-layer chromatography with ¹⁴C-labeled chloramphenicol. Signals were quantitated with a Molecular Dynamics PhosphorImager.

Transcriptional activities of different EBNA1 promoters in EBV(+) tumors and cell lines.

Transcripts (FUK, FUU′, QUK, and YUK) from the four different EBNA1 promoters (Fp, Qp, and Cp/Wp) could usually be amplified and distinguished by RT-PCR using five primers (F, Q, Y3, U′, and K) and a common probe as illustrated in Fig. 1A. In some instances additional primers, Q′ (upstream of Q) and U2′ (downstream of U′), were necessary. Analysis of 16 EBV-associated tumors, including tumors of lymphoid (B, T, and NK cell) or epithelial origin, showed that EBNA1 is consistently driven from Qp (Fig. 1B). Fp- or Cp/Wp-initiated EBNA1 transcripts were only occasionally detected in these tumors and when detected were always weak. Qp-, Fp-, and Cp/Wp-initiated transcripts were readily detected in most EBV(+) BL cell lines. However, type I BL cell lines (Rael, Akata, and Chep) lacked Cp/Wp-initiated EBNA1 transcripts (YUK), and two cell lines with integrated but not episomal viral genomes, Namalwa and IB4 (31), lacked lytic Fp-initiated transcripts (FUU′). These results are summarized in Table 2.

Methylation status of Qp.

CpG methylation has been implicated in the silencing of the EBV Cp in BL, HD, and NPC. The results above suggest that Qp is never silent. We carried out bisulfite genomic sequencing to determine whether it is ever methylated. The minimal Qp region and adjacent sequence include 20 CpG sites (Fig. 2A) (37, 39, 50, 63). All 16 CpG sites downstream of the Fp initiation site (which includes the entire Qp) were unmethylated in virtually all EBV(+) tumors and BL cell lines (Fig. 2C and 3). However, the four CpG sites upstream of the initiation site of Fp were variably methylated. In the Rael cell line, which is weak for Fp activity, these four CpG sites were partially methylated. 5-Azacytidine treatment leads to full demethylation of these CpG sites and increased Fp activity. In cell lines with strong lytic Fp activities, these four sites were always unmethylated.

Sequence conservation of Qp.

The sequence of Qp is highly conserved among specimens collected from Africa, North America, and Southeast Asia (Hong Kong and Taiwan). Only two instances of divergence from the prototype B95-8 sequence were found: a T-to-C mutation at 62403 in an African BL tumor (BL6) and a C-to-T mutation at 62275 in an Asian NTL isolate which abolishes CpG site 7 (Fig. 2C).

Effect of methylation on the transcriptional activity of Qp.

Qp appears to be consistently hypomethylated in the context of a viral genome that is generally hypermethylated. To evaluate whether the transcriptional activity of Qp is sensitive to methylation, we applied the methylation cassette assay (42). Recombinant plasmid pQpCAT, containing Qp from −172 to +32 (CpG sites 5 to 19) in a CAT reporter plasmid, was created. A cassette corresponding to Qp was excised and methylated in vitro with CpG methylase. The methylated cassette was ligated back into the CAT background, and the ligated mixture was transfected into the EBV(+) and EBV(−) BL cell lines Rael and CA46. In parallel, the unmethylated cassette was ligated back into the CAT background and also transfected. Methylation of the Qp fragment resulted in an average of 40.4- or 70.2-fold reduction in CAT activity in the Rael or CA46 cell line, respectively in three independent transfections (Fig. 4). Thus, the transcription of Qp is strongly inhibited by methylation and Qp is a methylation-sensitive promoter.