Identification of Sequences in Epstein-Barr Virus DNA Required for the Expression of the Second Epstein-Barr Virus-determined Nuclear Antigen in COS-1 Cells

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During latent Epstein-Barr virus (EBV) infection of human B lymphocytes, six viral nuclear antigens (EBNAs) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites. These transcripts initiate from one of two promoters, Cp or Wp, that function in a mutually exclusive fashion. Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes, followed by a switch to Cp usage. These studies have been extended to show that (i) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes, although the virus contains a functional Cp; (ii) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes, but only in the context of upstream and downstream sequences; (iii) this region contains an EBNA 2-dependent enhancer; and (iv) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer. These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2, followed by activation of Cp through the EBNA 2-dependent enhancer. Epstein-Barr virus (EBV) is

Section: Refs 1-3)mentioning

confidence: 99%

Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection.

Woisetschlaeger

Jin

Yandava

et al. 1991

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139

“…The pSVECAT plasmid, which carries the reporter CAT gene under the control of the simian virus 40 (SV40) early promoter, was obtained from M. Yaniv (Institute Pasteur, Paris). The EBNA2 expression vector pEAA6 and the mutated derivatives pEAA7, pEAA8, pEAA9, pEAA12, and pEAA13 with defined deletions in the EBNA2 gene are constructs based on the pSV2-gpt vector and have been described (19 (Fig. 1).…”

mentioning

confidence: 99%

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

Fåhræus

Jansson

Ricksten

et al. 1990

145

104

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...

“…A seeming paradox is that the coding capacity of BERF2a and BERF2b or BERF3 and BERF4 is only about 1000 amino acids, whereas the apparent molecular sizes of EBNA4 and EBNA6 determined by NaDodSO4/PAGE correspond to proteins of average composition containing about 1500 amino acid residues. Presumably, the discrepancies between the molecular weights predicted from the nucleotide sequence and the sizes deduced from the electrophoretic mobility reflect the unusual amino acid composition of these polypeptides, as has been observed for several other prolinerich proteins, including EBNA2 (11,13).…”

Section: Discussionmentioning

confidence: 92%

“…EBNA1 is encoded by a rightward open reading frame in the BamHI K fragment, BKRF1 (2)(3)(4)(5). EBNA2 is encoded by the BYRF1 reading frame in the BamHI Y and H fragment region (3,(6)(7)(8)(9)(10)(11)(12)(13). EBNA3 is largely encoded by the BERFM reading frame in the BamHI E fragment (14)(15)(16) and presumably the short BLRF3 frame in the BamHI L fragment (17).…”

mentioning

confidence: 99%

BamHI E region of the Epstein-Barr virus genome encodes three transformation-associated nuclear proteins.

Ricksten

Kallin

Alexander

et al. 1988

Recombinant vectors carrying DNA fragments from the Bamff E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the rmdings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.The Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) is a regularly detectable viral "footprint" in all EBV-transformed cells and is therefore believed to play an important role in growth transformation (1). The serologically defined EBNA has been dissected into several protein components. Five nuclear proteins present in latent infection have been identified, denoted EBNA1-EBNA5, and four of them have been localized on the genomic map. EBNA1 is encoded by a rightward open reading frame in the BamHI K fragment, BKRF1 (2-5). EBNA2 is encoded by the BYRF1 reading frame in the BamHI Y and H fragment region (3, 6-13). EBNA3 is largely encoded by the BERFM reading frame in the BamHI E fragment (14-16) and presumably the short BLRF3 frame in the BamHI L fragment (17 (20)(21)(22)(23)(24). It binds to specific sites in the oriP region of the EBV genome. This binding is essential for the maintenance and replication of episomal viral DNA. It may also influence the transcription of the viral genes expressed in growthtransformed cells. EBNA2 may be involved in the initial phase of B-cell transformation. This has been deduced from the fact that the P3HR-1 substrain of EBV, which lacks a large part of EBNA2, does not stimulate DNA synthesis in resting B lymphocytes and cannot transform them into immortal lines (25)(26)(27). Recent work has shown that EBNA2-expressing transfected Rat-1 cells have acquired the ability to grow at low serum concentrations (11) and that the growth properties of EBV-transformed lymphoblastoid cell lines (LCL) are influenced by the EBNA2 subtype of the resident virus (28). The function of the other members of the EBNA family is unknown.In the present report we show that the larger of the two polypeptides d...