The 5′ flanking region of the gene for the Epstein-Barr virus-encoded nuclear antigen 2 contains a cell type specificcis-acting regulatory element that activates transcription in transfected B-cells

Abstract

We have recently identified the promoter that positions the initiation (cap) site for RNA encoding the Epstein-Barr virus (EBV) determined nuclear antigen 2 (EBNA2) in transfected COS-1 cells. The cells were transfected with recombinant vectors that contained the BamHI WYH region of the EBV genome. In order to delineate regulatory DNA sequences required for the expression of EBNA2 the 5' flanking region of the gene was linked to reporter genes in expression vectors and transfected into EBV genome-negative lymphoid DG75 cells. We demonstrate that several cis-acting elements contribute to a transcriptional enhancer activity found in the region between nucleotides-553 and -86 relative to the cap site. The enhancer was active in lymphoid DG75 cells but not in HeLa cells and stimulated transcription also from the heterologous thymidine kinase (TK) and beta-globin promoters. Nuclear extracts of lymphoid cells contained protein factors that bound to the enhancer. The in vitro introduction of a mutation in the enhancer sequence that substantially reduced the transcription stimulatory activity concurrently blocked the binding of one of the factors.

Host cell phenotype-dependent methylation patterns of Epstein--Barr virus DNA

“…Thick lines indicate important coding or regulatory sequences within the regions probed. The following probes were used: (i) an EcoRI-BamHI subfragment from the BamHI C fragment (nucleotides 7315 to 13 215) covering oriP, the latent origion of EBV replication (Yates et al, 1984); (ii) the BamHI W fragment containing a B cell-specific'enhancer (W ENH) (Ricksten et al, 1988); (iii) the BamHI H fragment partly covering EBNA 2 coding sequences and containing oriLyt, the lytic origin of EBV replication (Hammerschmidt & Sugden, 1988); (iv) the BamHI M fragment containing coding sequences for early antigens (open reading frames BMRF 1 and BMRF 2); (v) the BamHI E fragment encoding EBNA 3, 4 and 6; (vi) the BamHI K fragment.encoding EBNA l ; (vii) a 2.4 kb MluI subfragment of the EcoDHET fragment covering the coding sequences of LMP (nueleotides 167129 to 169 566); (viii) a 0.8 kb BglI fragment (nucleotides 169 449 to 170290); or (ix) a XhoI-EcoRI fragment (nucleotides 169 424 to 172 284) for the analysis of regulatory sequences (LRS) of the LMP gene (F~thraeus et al, 1990). (ii) Immunoblotting.…”

Epstein–Barr virus U leader exon contains an internal ribosome entry site

“…Cells were harvested 48 h post-transfection and aliquots of cell lysates were assayed for LUC and CAT activities. The method for measuring CAT activity has been described previously (Ricksten et al, 1988). The LUC activity was determined with the Luciferase Assay System (Promega) using a TD 20/20 luminometer (Turner Designs Instruments, USA).…”

“…The expression of EBNA1 is controlled at multiple levels. The transcriptional regulation of EBNA1 involves initiation from three alternative promoters, Wp (Sample et al, 1986;Ricksten et al, 1988), Cp (Woisetschlaeger et al, 1990) and Qp (Schaefer et al, 1995b;Tsai et al, 1995), which are used differentially during different phases of infection and establishment of the stages of latency. During the viral lytic cycle, EBNA1 mRNA is transcribed from a fourth promoter called the Fp promoter (Schaefer et al, 1995a;Nonkwelo et al, 1996).…”

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.

“…For the synthesis of $#P-labelled RNA as hybridization probes, plasmid DNA was transcribed in vitro in the presence of [α-$#P]CTP (3000 Ci\mmol ; DuPont NEN) by a standard procedure (Sambrook et al, 1989). Full-length RNA molecules were purified by PAGE and used as probes in the RNase protection assay (Ricksten et al, 1988) with the following modifications : 50 µg DNasetreated cytoplasmic RNA was mixed with $#P-labelled RNA (1i10' c.p.m.) and incubated at 50 mC for 16 h. Single-stranded material was hydrolysed by the addition of 300 µl digestion mixture (10 µg\ml RNase A, 16 U\ml RNase T1, 0n30 M sodium acetate, pH 7n0, 5n0 mM EDTA) and incubation at 37 mC for 60 min.…”