Host-cell-phenotype-dependent control of the BCR2/BWR1 promoter complex regulates the expression of Epstein-Barr virus nuclear antigens 2-6.

Abstract

(7). This is reflected by the decrease or disappearance of BL-associated CD10 and CD77 markers and the appearance of activation markers. EBNAs 2-6 and LMPs 1, 2a, and 2b are upregulated as well. Nasopharyngeal carcinoma (NPC) represents yet a third type of EBV-host cell interaction. EBNA 1 is always expressed but EBNAs 2-6 are not. LMP 1 is detected in some 65% of NPCs (9).We have found (10, 11) that somatic cell hybrids derived from the fusion of EBV-carrying group III BL lines or LCLs with non-B-cell lines downregulate EBNAs 2-6, LMP 1, and B-cell-specific markers. This suggested that the regulation of EBNA 1, on the one hand, and EBNAs 2-6, on the other hand, can be dissociated from each other. EBNA 1 shows a dominant constitutive expression, and EBNAs 2-6, LMPs, and B-blast-specific markers are expressed in a cellphenotype-dependent fashion. The purpose of the present study was to examine whether the EBNA 2-6 expression depends on selective promoter activity. Our S1 nuclease analysis has shown that group I BL cells, NPC biopsies, and somatic cell hybrids that express only EBNA 1 utilize neither the BCR2 nor the BWR1 promoter. As our earlier findings (9,12,13) suggested that viral DNA methylation may play a role in the regulation, we investigated whether promoter activity correlated with the methylation status. MATERIALS AND METHODSCells. The origin and EBV gene expression of all the cell lines is summarized in Table 1 The biopsies were stored frozen at -70'C until used for RNA isolation. C15, CAO, and NPCs 1 and 3 were LMP-positive whereas NPCs 2 and 4 were negative by immunoblot analysis with the S12 monoclonal antibody. They were all EBNA 1-positive (9).RNA Preparation and S1 Nuclease Assay. Total RNA from cell lines was prepared by homogenization in guanidinium isothiocyanate and ultracentrifugation through a CsCl cushion. Total RNA from NPC material was prepared by acidguanidinium thiocyanate/phenol/chloroform extraction.

Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

“…FpQUK(557) constituted 1-6 % of the Kcontaining transcripts in induced cell lines (Table 2). RT-PCR with primers Q1 and K2 generated an amplification product that hybridized to the U and K probes, reflecting this small proportion of Fp-initiated transcripts spliced to the EBNA1-specific splice acceptor site in BamHI K. Induction of Rael cells with 5-azacytidine is known to up-regulate Cp\Wp-initiated transcription (Altiok et al, 1992 ;Jansson et al, 1992), shown here by the increased amounts of the protected UK(332) and U(172) fragments and the appearance of a specific amplification transcript that is initiated upstream of the BamHI f/K cleavage site, passing through BamHI K directly into the EBNA1-encoding K exon. The K(350-370) fragments represent transcripts that contain the part of BamHI K upstream of the splice acceptor site for BKRF1, most likely corresponding to a lytic BRRF2 transcript.…”

Constitutive Activation of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Gene Transcription by IRF1 and IRF2 during Restricted EBV Latency

“…A number of studies have shown that the type III latency EBNA gene promoters, Cp and Wp, are inactivated by CpG methylation during type I latency (2,10,22,44,49,62,69), while Qp is at the center of a hypomethylated island and remains active (73). Although evidence strongly suggests that methylation of Cp and Wp is absolutely required for EBV to stably maintain the type I and II latency programs (73), almost nothing is known about the specific trans-acting factors which regulate transcription from Qp.…”

Latency type-specific distribution of epigenetic marks at the alternative promoters Cp and Qp of Epstein–Barr virus

“…This discrepancy may be due to the different passage history of the cells used. While Cp activity is regulated by both CpG methylation (Altiok et al, 1992;Minarovits et al, 1994;Robertson et al, 1995;Salamon et al, 2001;Bakos et al, 2007) and histone modifications (this study), Qp is unmethylated independently of its activity (Tao et al, 1998;Salamon et al, 2001). We observed that similar to active Cp, active Q promoters in lymphoid and epithelial cells are also located on an acetylation island enriched in AcH3 and AcH4.…”

“…Mutu-BL-I-Cl-216 maintains the phenotype of BL biopsy cells and expresses EBNA 1 and the small EBV-encoded RNAs, EBER 1 and 2, but EBNA 2-5 and latent membrane proteins (LMPs) cannot be detected in this clone (type I latency, Cp off, Qp on). In contrast, Mutu-BL-III-Cl-99 has a lymphoblastoid phenotype and expresses all six EBNAs, EBER 1 and 2, and LMP 1 and 2 (type III latency, Cp on, Qp off) (Gregory et al, 1990;Altiok et al, 1992;Bakos et al, 2007). The BL line Rael (latency I) and the lymphoblastoid cell line (LCL) CB-M1-Ral-STO (latency III) also carry the same EBV strain (Ernberg et al, 1989).…”

“…1f). Cp was active only in type III latency, as described previously (Woisetschlaeger et al, 1991;Altiok et al, 1992;reviewed by Györy & Minarovits, 2005). TSA alone or combined with cycloheximide (CHX) induced Cp-initiated transcription in Mutu-BL-I-Cl-216, although this induced transcription did not reach the level of Cp activity detected in the latency type III Mutu clone (Weinmann & Farnham, 2002) was prepared from the indicated cells and immunoprecipitated with specific antibodies (Upstate) directed to diacetylated histone H3 (a), tetraacetylated histone H4 (b), histone H3 dimethylated at lysine 4 (H3K4me2) (f) or was mock-precipitated with a non-specific antibody (c).…”