Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals

Abstract

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.

FIG. 1

Schematic illustration of EBV EBNA gene transcription during immortalizing (type III) and restricted (types I and II) viral latency. Shown is a linear representation of the EBV genome, with the locations of the EBNA gene promoters Cp, Wp, and Qp indicated. The general organization of exons present in EBNA gene transcripts during immortalizing (type III) latency and restricted (type I and type II) latency is shown above the viral genome. Type III latency involves expression of all six EBNAs, generated by alternative splicing of long primary transcripts that initiate at either Cp or Wp. During type I or type II latency, only one of the EBNAs, EBNA 1, is expressed, and its transcription is driven from Qp (Cp and Wp are silent during restricted EBV latency). Fp is a lytic promoter located ca. 200 bp upstream of Qp (34, 64); oriP is the latency-associated origin of episomal replication; IR1, the major internal repeat, consists of 8 to 10 copies of the 3.0 kb BamHI W fragment. Note that the sizes of the small 5′ EBNA gene exons are exaggerated for the sake of clarity. EBNA 1 binding to oriP functions to upregulate Cp and Wp activity, as well as to assist with maintenance of the viral episome. EBNA 1 also functions to downregulate Qp through binding to two low-affinity sites downstream of Qp. TR, direct terminal repeats present at each end of the linear viral genome which mediate circularization of the viral genome upon infection.

FIG. 2

In vivo methylation status of the EBV EBNA gene promoters Wp, Cp, and Qp in peripheral B cells. The genome coordinate for each CpG within the regions examined is given at the left. ●, methylated CpGs; ○, unmethylated CpGs. Each numbered column represents the data obtained from an independent bisulfite PCR reaction. Genomic DNA was isolated from the B cells of three healthy EBV-seropositive human donors (donors 8, 9, and 10) and from the EBV-negative BL cell line DG75 (negative control DNA). DNA was bisulfite treated and amplified by nested PCR. PCR products were cloned and sequenced. Conversion of non-CpG cytosines into thymines was at least 99.6% complete for all PCRs analyzed. For the compiled data shown, no false positives were detected in 50 control PCRs carried out with DG75 EBV-negative DNA and 93 water control PCRs. (A) CpG methylation in the region upstream of Cp. Eighteen independently generated bisulfite PCR products were analyzed. Also shown are the locations of the EBNA 2 enhancer (EBNA2-enh), the CCAAT boxes, and the Cp TATAA box and transcription start site. The P at genome coordinate 11210 in PCR 5 from donor 8 represents a polymorphism (CpG-to-TpG mutation). (B) CpG methylation in the region upstream of Wp. The analysis of 18 independently generated bisulfite PCR products is shown. The Cp/Wp enhancer, the CCAAT box, the Wp TATAA box and transcription start site, the W0 exon, and the W1 exon are indicated as well. (C) Hypomethylation of the region immediately upstream of Qp and hypermethylation of the region upstream of Fp. Sixteen independently generated bisulfite PCR products were analyzed. Also shown are the locations of the Fp TATAA box and transcription start site, the IRF1/IRF2 binding site, the Qp transcription start site, and the low-affinity EBNA 1 binding sites.

FIG. 3

Summary of the methylation status of the EBNA gene promoters in the long-term latency reservoir in healthy seropositive individuals. The diagram schematically illustrates EBNA 1 gene transcription from Qp and also depicts EBNA 1 inhibition of Qp-initiated transcription. Notation is as for Fig. 1.

FIG. 4

Cp-initiated transcription from a transiently transfected reporter construct in a BL cell line exhibiting restricted EBV latency. Rael, an EBV-positive BL cell line that displays restricted (type I) latency, was transfected with 10 μg of either the CW1CAT reporter plasmid (Fig. 4A) or an irrelevant luciferase (control [cntl]) plasmid. Total RNA was isolated at 72 h posttransfection, and the presence of Cp- and Wp-initiated transcripts was assessed by S1 nuclease protection. RNA was hybridized with ³²P-labeled Cp, Wp, or β-actin S1 probes. RNA samples from the EBV-infected lymphoblastoid cell line (LCL) JY, which drives EBNA gene transcription from Cp, and from the BL cell line Clone-16 (Cl-16), which initiates EBNA gene transcription at Wp, served as positive controls for Cp and Wp activity. Identical results were obtained in two independent experiments.

FIG. 5

Hypermethylation of the region upstream of Wp in the CW1CAT reporter plasmid inhibits Cp-driven CAT activity. (A) Outline of the methylation cassette assay. A diagram of the CW1CAT plasmid, showing the locations of Cp, Wp, and relevant restriction endonuclease digestion sites (B, BamHI; H, HindIII) is shown. FR, family of repeats; DS, dyad symmetry element; C1 and C2, first two exons of Cp-initiated transcripts. (B) Southern blot demonstrating that methylated and unmethylated W fragments ligate into the CW1CAT vector with equal efficiency. The methylation cassette assay was performed as outlined in panel A. Diagnostic digestion of the W inserts with the methylation-sensitive restriction endonuclease SmaI demonstrated that the methylated W insert was completely resistant to digestion, while the unmethylated insert could be completely digested (data not shown; the W insert contains six SmaI cleavage sites). The ligations were carried out as described in Materials and Methods. Prior to transfection, a small aliquot of each ligation was removed, digested with either BglII/XbaI or EcoRI, and probed with the ³²P-labeled BglII/HindIII fragment isolated from the CW1CAT construct. CW1CAT plasmid digested with BglII/XbaI or EcoRI served as a positive control (cntl). The 2,243-bp band in the BglII/XbaI digest and the 7,438-bp band in the EcoRI digest both represent correctly ligated DNA. The 1,540-bp band in the EcoRI digest represents inserts ligated only to the HindIII site of the vector. PhosphorImager (Molecular Dy- namics) quantitation demonstrated that there was no apparent difference between the ligation efficiencies of methylated and unmethylated DNA (the additional bands represent other minor ligation species that make up less than 10% of the total DNA in the ligation, as determined by PhosphorImager quantitation). (C) CAT activity recovered from cells transfected with either the unmethylated W, methylated W, or vector control ligations. The ligations were transfected into Rael cells, and CAT assays were carried out at 72 h posttransfection. The unacetylated and monoacetylated species of [¹⁴C]chloramphenicol were quantitated by PhosphorImager analysis, and the percentage of acetylated chloramphenicol is given below each lane. The experiment shown is representative of four independent experiments, and the observed decrease in CAT activity upon methylation of the W insert ranged from 8- to 16-fold.