An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators

Abstract

The EAR and NotI repeat genes of Epstein-Barr virus (EBV) are oriented head to head and separated by a 1,000-base-pair (bp) divergent promoter region. We have identified functional domains within this divergent promoter which are important for regulation of the rightward EAR gene. Both the R transactivator (Rta) and the Z transactivator (Zta) increase the abundance of correctly initiated EAR transcripts. A 258-bp fragment (-114 to-372 from the EAR cap site) contained the primary Rta and Zta response elements and was capable of transferring Rta and Zta activity to a heterologous promoter in an orientationand position-independent manner. Rta activated this 258-bp enhancer region in both EBV-positive and EBV-negative cells. However, Zta activity appeared to be dependent on another EBV gene product, since Zta activated the enhancer efficiently (500to 2,000-fold) in EBV-positive cells but had little or no activity in EBV-negative cells. The combination of Rta and Zta produced a striking synergistic effect on the enhancer in the absence of any additional EBV components, suggesting that the interaction between Zta and Rta accounts for the Zta response observed in EBV-positive cells. An Rta response element was mapped to a domain located 60 bp away from a Zta-binding site within the enhancer. Although Rta activated the enhancer and other early promoters without additional EBVor B-cell-specific factors, it did not activate the lytic cycle of EBV, in contrast to Zta. Immunofluorescence patterns of Rta and Zta with antipeptide antisera indicated that they have overlapping but different subcellular localizations. Both transactivators were found in the nucleus, but Rta was also found in the cytoplasm.

Short‐term assays for detection of conditional cancerogens I. Construction of DR‐CAT Raji cells and some of their characteristics as tester cells

“…The failure to induce DR-CAT in EBV-negative cells is in line with the finding that BZLFl protein is required as a transcriptional activator to switch on other early viral genes, including the DR gene. A number of BZLFI-protein-binding sites have been identified within the DR promoter (Chavrier et al, 1989;Chevallier-Greco et al, 1989;Lieberman et al, 1989;Cox et al, 1990;Gruffat et al, 1990).…”

Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays

“…Rta can contact both TBP and TFIIB (53). Zta and Rta in combination produce a synergistic response in targets containing binding sites for both factors (14,68). The third transactivator, Mta (encoded by BSLF2/BMLF1), has a posttranscriptional mechanism of action (4,45).…”

“…Deletion of the TATA box, ZRE1, ZRE2, and the CAAT box abolishes replication, while mutation of the ZRE1 and ZRE2 sites reduces replication efficiency fourfold (73). The enhancer contains binding sites for both Zta and Rta and responds synergistically to the presence of these transactivators (14). Mutation of the Zta binding sites in the enhancer in conjunction with mutation of the four promoter Zta binding sites ablates replication ability, providing evidence for an essential role for Zta in lytic replication (72).…”

Regulation of the herpesvirus saimiri (HVS) delayed-early 110-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R trans activator

“…In the alignments, similar residues are indicated by a single dot or a pair of dots (joining well-conserved amino acids) and identical residues are indicated by vertical lines. product has been defined as the initial trigger for the lytic cycle (10,39,40,42), but its function is dependent on the presence of the BRLF1-encoded protein (9,12,24). We predicted that the homologs of these genes in HVS, if they were present, would occur within the EcoRI-D (15-kbp) fragment of HVS L-DNA (Fig.…”