Transcriptional regulation of the Epstein-Barr virus nuclear antigen 1 gene in latent and lytic stages of infection

Abstract

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus that infects more than 90% of humans worldwide. The virus is the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disorder, and is also associated with a variety of malignancies including Burkitt's lymphoma (BL), Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), and lymphoproliferative disorders in immunocompromised individuals. EBV can adopt four distinct programs of latency (latency 0, I, II and III), in which different combinations of the EBV gene products (EBV nuclear antigens; EBNA1-6, and latent membrane proteins; LMP1, LMP2A and LMP2B) are detected. In all latency types where the virus genome is maintained within perpetually dividing cells the EBNA1 gene is expressed. EBNA1 is essential for replication and partitioning of the virus genome during multiple cell divisions and is thus fundamental to the tumour-causing capacity of EBV. The aim of the present investigation was to define cellular and viral controls of EBNA1 gene expression. Four promoters direct EBNA1 gene transcription, Wp, Cp, Fp and Qp. The promoters are differentially used during the different phases of virus infection. By RNase protection and RT-PCR analyses we show that Wp and Cp are predominantly used in latency III, whereas Qp is the EBNA1-selective latency I and II promoter. Fp is down-regulated in latently infected cells both in the endogenous virus genome and in reporter plasmids and is activated upon induction of the virus lytic cycle. Thus, Fp is a lytic and not a latent promoter. Only a minority of the Fp-initiated transcripts are actually spliced to the regular EBNA1-splice acceptor in the EBNA1-encoding exon. The repression of Fp in latent stages of infection can be abolished by the main initiator of the virus lytic programme, the virus lytic switch protein ZEBRA. By electrophoretic mobility shift assay (EMSA) and transient transfection experiments, we demonstrate that ZEBRA activates Fp through a direct interaction with an AP-1-like site at position -52/-46 in the promoter-proximal Fp region. Deletion and mutation analyses of Cp reporter plasmids in combination with EMSA show that the latency III C promoter is controlled by several cellular transcription factors in addition to the viral proteins EBNA1 and EBNA2 that are known to transactivate Cp. The NF-Y transcription factor interacts with a CCAAT box at position -71/-63 in Cp. Sp1 and Egr-1 interact with a GC-rich sequence at position -99/-91 in Cp. Members of the C/EBP transcription factor family interact with a C/EBP-like site at position -119/-112 in Cp. NF-Y, Sp1 and C/EBP are positive regulators of Cp activity, whereas the functional role of Egr-1 remains to be established. Sp1 and NF-Y activate Cp synergistically and both are required at the promoter for a significant transcription to occur. The biological significance of NF-Y is further emphasised by the finding that NF-Y expression is highly correlated to endogenous Cp activity in B-lymphoid cells. In summary, EBNA1 gene transcription is regulated in a highly complicated manner. Both viral and cellular transcription factors control the different EBNA1 gene transcription units, but the regulation is remarkably dependent on host cell factors.