Interferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma.

Replication of the Epstein-Barr virus (EBV) genome within latently infected cells is dependent on the EBV EBNA-1 protein. The objective of this study was to identify transcriptional regulatory proteins that mediate EBNA-1 expression via the viral promoter Qp, which is active in EBV-associated tumors such as Burkitt lymphoma and nasopharyngeal carcinoma. Results of a yeast one-hybrid screen suggested that a subset of the interferon regulatory factor (IRF) family may regulate EBNA-1 transcription by targeting an essential cis-regulatory element of Qp, QRE-2. Further investigation indicated that the transcriptional activator IRF-1 and the closely related IRF-2, a repressor of interferon-induced gene expression, are both capable of activating Qp. However, the major QRE-2-specific binding activity detected within extracts of Burkitt lymphoma cells was attributed to IRF-2, suggesting that interferon-independent activation of Qp is largely mediated by IRF-2 in these cells. We observed no effect of gamma interferon on Qp activity in transfection assays, whereas we observed a moderate but significant repression of Qp activity in response to alpha interferon, possibly mediated by either the interferon consensus sequence binding protein or IRF-7, a novel alpha interferon-inducible factor identified in this study. Since expression of IRF-1 and IRF-2 is increased in response to interferons, the Qp activity observed in the presence of interferon likely represented an equilibrium between IRF factors that activate and those that repress gene expression in response to interferon. Thus, by usurping both IRF-1 and its transcriptional antagonist IRF-2 to activate Qp, EBV has evolved not only a mechanism to constitutively express EBNA-1 but also one which may sustain EBNA-1 expression in the face of the antiviral effects of interferon.

The Epstein-Barr virus EBNA-1 promoter Qp requires an initiator-like element.

Expression of the Epstein-Barr virus (EBV) EBNA-1 protein within EBV-positive tumor cells and subpopulations of latently infected B lymphocytes in vivo is mediated by the promoter Qp. Previous studies have established that Qp is a TATA-less promoter whose activation requires only proximal regulatory elements and that it is negatively autoregulated through two EBNA-1 binding sites downstream of the transcription initiation sites. The objective of this study was to better define the properties of an essential positive regulatory element (QRE-2) adjacent to a major transcription start site of Qp and to evaluate the contributions of other potential regulatory elements proximal to the Qp start site. Using DNA affinity purification and UV cross-linking, we have identified the QRE-2-binding protein as a single polypeptide of approximately 40 kDa. The DNA-binding properties of this protein are clearly distinct from those of the TATA-binding protein, suggesting that in the absence of a TATA box, QRE-2 may function as an initiator element to direct assembly of TFIID near the transcription start site. Mutational analysis of potential regulatory elements, furthermore, indicated that the putative E2F binding sites within the EBNA-1 binding domain can exert a positive influence on Qp that is EBNA-1 independent, suggesting that these regulatory elements play an additional if not different role in Qp regulation than previously proposed. A model for the regulation of Qp consistent with the current and previous findings which provides for a simple but efficient mechanism of ensuring the EBNA-1 expression necessary to sustain long-term latency is presented.

Transcription start sites downstream of the Epstein-Barr virus (EBV) Fp promoter in early-passage Burkitt lymphoma cells define a fourth promoter for expression of the EBV EBNA-1 protein.

In Epstein-Barr virus (EBV)-transformed B lymphoblastoid and many Burkitt lymphoma cell lines, the EBV EBNA-1 protein is one of six viral nuclear antigens expressed from a common transcription unit under the control of one of two promoters, Wp or Cp. In contrast, EBNA-1 is the only EBV nuclear antigen expressed in Burkitt and other EBV-positive tumors. We previously identified a promoter of EBNA-1 transcription, designated Fp, in early-passage Mutu Burkitt tumor cells, and this promoter is also active in long-term Mutu and Akata Burkitt cell lines which maintain the exclusive expression of EBNA-1 characteristic of the tumor. However, transcription initiation within Fp reporter gene plasmids in EBV-negative cells occurs at positions 100 to 200 bases downstream of the Fp start site in the BamHI-Q restriction fragment. Here we demonstrate that transcription initiation within newly established Burkitt lymphoma cell lines is consistent with the transcription initiation we observed in reporter plasmids. Furthermore, previous observations of transcription from Fp to generate EBNA-1 transcripts can be attributed to lytic-cycle gene expression. These data, in conjunction with our previous characterization of promoter regulatory elements, define a fourth EBNA-1 promoter, Qp, that is active in latently infected Burkitt tumor cells.

Characterization of the Epstein-Barr virus Fp promoter.

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) protein is mediated by the virus Fp promoter in Burkitt lymphoma and nasopharyngeal carcinoma. This promoter is silent in latently infected B lymphoblastoid and most Burkitt lymphoma-derived cell lines in vitro, which utilize separate promoters approximately 50 kb upstream of Fp to express EBNA proteins. Fp-mediated activation of EBNA-1 expression is also activated upon induction of the virus replication cycle. We previously demonstrated that activation of Fp in Burkitt cells requires cis-regulatory elements downstream of the site of transcription initiation. We have now mapped two positive regulatory elements within the Fp promoter. One element contains two potential binding sites for the cellular transcription factor LBP-1 between +138 and +150. A second regulatory element was mapped between +177 and +192 and can be specifically bound in vitro by protein from nuclear extracts of Burkitt cells. Although this element overlaps two partial E2F binding sites and Fp reporter plasmids could be activated in trans by the adenovirus E1A protein in cotransfection experiments, mutational analysis and DNA binding studies suggest that these are unlikely to be functional E2F response elements within Fp. We also demonstrate that Fp-directed transcription initiates at multiple sites within both the genome and the Fp reporter plasmids. However, the principal site of transcription initiation within the genome is not utilized within reporter plasmids, in which the majority of transcripts initiate at multiple sites between +150 and +200. This finding suggests that additional elements may be necessary for Fp to function normally in these assays or that the context of Fp within the viral genome is critical to its regulation.

Regulation of Epstein-Barr virus BamHI-H divergent promoter by DNA methylation.

The regulatory mechanisms which control latency and reactivation of the Epstein-Barr virus (EBV) are not fully understood. To determine whether DNA methylation is involved, we examined the BamHI-H divergent promoter, which also encompasses the origin for lytic replication (oriLyt) of EBV. The divergent promoter was highly methylated in the stringently latent HH514cl16 cell line and largely unmethylated in the semipermissive FF41-1 cell line. Expression vectors in which the divergent promoter directed transcription of the chloramphenicol acetyltransferase gene were made. Using in vitro methylation and transient transfections, we found an inverse correlation between the number of sites methylated and level of gene expression. Similar patterns of inhibition were observed when the methylated promoter was activated by BZLF1 or BRLF1 and in lymphoid or epithelial cells. The role of two CpG dinucleotides in the BRLF1 binding sites of the divergent promoter was determined by site-directed mutagenesis. The results indicated that site-specific methylation of these CpGs was not solely responsible for inhibition of expression by methylation. DNA methylation also reduced DNA replication mediated by oriLyt. These results suggest that hypermethylation of the divergent promoter and oriLyt may suppress transcription and lytic replication of EBV.