Epstein-Barr virus-encoded nuclear antigen 2 activates the viral latent membrane protein promoter by modulating the activity of a negative regulatory element.

Abstract

Previous studies suggest that the EpsteinBarr virus nuclear antigen EBNA2 participates in the regulation of the expression of the viral latent membrane protein (LMP). We have used reporter plasmids containing DNA fragments of the 5' flanking region of the LMP gene in cotransfection experiments to analyze the effect of EBNA2 on the activity of the LMP promoter. The results show that the LMP promoter is controlled by positive and negative transcription elements in a DNA fragment that contains the LMP transcription initiation site and 634 base pairs of upstream sequences. The promoter is activated by EBNA2. The region between position -54 and +40 relative to the mRNA cap site contains a positive transcription element that is constitutively active in DG75 cells and independent of EBNA2. The -106 to -54 region contains a negative regulatory element that prevents adjacent positive elements from functioning in the absence of EBNA2. Regulatory sequences between -324 and -144 participate in maintaining a high level of transcription of the LMP promoter after induction with EBNA2. The regulatory elements in the -634 to -54 promoter region have the characteristics of an inducible enhancer, including orientation independence and ability to regulate a heterologous promoter. In this context it might be relevant that transfected EBNA2 could induce the expression of the activation antigen CD23 on the surface of an EBV-negative Burkitt lymphoma (BL) cell line (6). The CD23 antigen is related, to the receptor for a B-cell growth factor (7) and has also been suggested to act as an autocrine growth factor for B cells after shedding from the cell surface (8). Epstein-Barr virus (EBVEBNA2 is a phosphorylated polypeptide with DNAbinding properties, which is encoded by the BYRF1 reading frame of the BamHI WYH region of the EBV genome (reviewed in ref. 1). The protein (the A subtype) has a rather unusual primary structure containing an almost continuous sequence of 40 proline residues, an arginine-and glycine-rich positively charged region, and a negatively charged Cterminal sequence. The overall proline content of the 487-amino acid long polypeptide is 29%. The recent demonstration of a new class of transcriptional activators with a proline-rich domain might provide some clues concerning the action of EBNA2 (9).It is thus conceivable that EBNA2 modulates the cell phenotype by influencing the expression of cellular genes. The effect might be direct, as suggested by the induction of CD23 in EBNA2-transfected cells (6), or indirect and mediated by way of other EBV genes, whose expression is in turn controlled by EBNA2. Evidence for the latter mechanism has been provided recently. The B95-8 virus strain, but not P3HR-1, has been shown to be able to induce expression of LMP in EBV-negative BL cell lines (10). Transfection of the EBNA2 gene into P3HR-1 virus-converted B-lymphoma cell lines induced the expression of LMP and a dramatic change in growth phenotype toward a LCL-like pattern (11). Some, but not all, of these changes could b...

Epstein--Barr virus (EBV) nuclear antigen 6 induces expression of the EBV latent membrane protein and an activated phenotype in Raji cells

“…An alternative, and perhaps more likely, explanation of these data is that EBNA 6 in some way augments the action of, or cooperates with, EBNA 2 in increasing the expression of LMP. EBNA 2 activates the LMP gene by inducing transcription through an EBNA 2-responsive element located approximately 50 to 100 bp upstream of the transcriptional initiation site (Fahraeus et al, 1990;Wang et al, 1990b). EBNA 2 protein does not appear to bind directly to the DNA at this site and may act by counteracting the effects of a cellular repressor protein (Fahraeus et al, 1990).…”

“…EBNA 2 activates the LMP gene by inducing transcription through an EBNA 2-responsive element located approximately 50 to 100 bp upstream of the transcriptional initiation site (Fahraeus et al, 1990;Wang et al, 1990b). EBNA 2 protein does not appear to bind directly to the DNA at this site and may act by counteracting the effects of a cellular repressor protein (Fahraeus et al, 1990). At this stage we cannot exclude the possibility that EBNA 6 acts indirectly through some undefined cellular pathway or even acts post-transcriptionally.…”

“…In addition EBNA 2 is a transcription factor which can activate the expression of LMP (Fahraeus et al, 1990;Wang e t al., 1990 b) and, by acting on the major rightward promoter (Cp or BCR 2) in the BamHI C region of EBV (Sung et al, 1991;Woisetschlaeger e t al., 1991), may also initiate or enhance the expression of EBNAs 1, 3, 4 and 6 (Woisetschlaeger et al, 1991). It is not surprising, therefore, that EBNA 2 has been shown to be essential for immortalization .…”

Antiviral agent Cidofovir decreases Epstein–Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies

“…It has been shown that EBNA2 and EBNA leader protein (EBNA-LP) cooperate to induce the transition of the infected cells from G0 to G1 (Sinclair et al, 1994). EBNA2 also activates the transcription of all viral proteins expressed in lymphoblastoid cell lines by transactivating: (i) the BamH1-C-promoter (Cp) (Sung et al, 1991), from which transcription of all EBNA genes is controlled; (ii) the promoters of LMP1 and LMP2 (Abbot et al, 1990;Fahraeus et al, 1990). Oncogenic potential of LMP1, frequently expressed in several EBV-associated cancers, is consistent with its ability to transform fibroblasts and to inhibit epithelial differentiation (Dawson et al, 1990).…”

Domains of the Epstein--Barr virus nuclear antigen 2 (EBNA2) involved in the transactivation of the latent membrane protein 1 and the EBNA Cp promoters

“…We and others have demonstrated that the regulatory region of the LMP1 EBV gene contains cis-acting elements that mediate the EBNA2-induced up-regulation of LMP1 expression (FAhraeus et al, 1990;Ghosh & Kieff, 1990). To define further the LRS elements involved in interactions with transcription factors in B cells differing in EBNA2 expression, DNase footprinting experiments were performed.…”