Regulation of the Epstein-Barr virus C promoter by the OriP-EBNA1 complex

Abstract

Epstein-Barr Virus (EBV) is an exclusively human, lymphotropic herpes virus that infects more than 90% of the population worldwide. Primary infection usually occurs during the early years of life and does not result in any recognized disease. EBV is the causative agent of infectious mononucleosis and is associated with various malignancies including Burkitt’s lymphoma (BL), Hodgkin’s disease (HD), nasopharyngeal carcinoma (NPC), and immunoblastic lymphomas in immunocompromised individuals. In most immunocompetent individuals the virus is, however, harbored for life within latently infected resting memory B cells, causing no symptoms. In vitro, EBV efficiently transforms resting B cells to activated lymphoblasts. These perpetually dividing cells express a repertoire of viral antigens (EBNA1-6 and LMP1), all of which have been directly implicated in the immortalization process. Immediately postinfection, EBNA2 and -5 are the expressed from the W promoter (Wp), within 36 hours there is a switch in promoter usage from Wp to the upstream C promoter (Cp). Transcription from Cp leads to a concomitant expression of all EBNAs from a polycistronic transcription unit that is spliced to yield the different EBNA proteins. EBNA1 forms multiple homodimers that bind to a portion of the latency origin of replication (oriPI) that functions as an EBNA1- dependent enhancer of the Cp. The mechanism for the interaction between the oriPI-EBNA1 complex and the Cp is not completely understood at the molecular level. EBNA1 has no apparent enzymatic activities and is thought to fulfill its functions by mediating interactions with specific host cellular proteins, only few of which have been characterized. The aim of this thesis was to identify and characterize the interaction partners of this macromolecular complex. The interactions of the transcription factors NF-Y and Sp1 with the promoterproximal region of the Cp were previously established in our lab. In paper I we studied these interactions further using transient transfections, establishing that NF-Y and Sp1 co-stimulate Cp and that the oriPI-EBNA1-induced transactivation of Cp requires concomitant expression of both proteins. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrated that inactivation of EBNA2 resulted in decreased expression of NF-Y and down-regulation of Cp. Knowing that resting B cells do not express NF-Y and observing that this factor is essential for Cp activation, we suggest that its up-regulation post-infection may contribute to the Wp-to-Cp switch in primary EBV infection. The oriPI contains 20 repeats of the EBNA1 binding domain. In paper II we used a series of oriPIdeletions in oriPI-CpCAT reporter plasmids in transient transfections to determine the number of EBNA1 binding repeats necessary for efficient transactivation of the Cp. We showed that eight or more repeats were necessary for this effect, which underscores the complexity of the transactivation process. In papers III and IV we set out to identify novel interaction partners of the oriPIand -170Cp regions using EMSA and DNA affinity purification coupled with mass spectrometry. Three novel protein interactions with the oriPI and the Cp were identified. The transcription factors Bright, E2F1 and Oct-2 were found to bind both sequences in vitro and in vivo, opening up a possibility of mediating a link between the oriPI and the Cp. The binding sites of all three proteins were mapped to a short segment of Cp in close proximity of each other. This region was previously shown to be essential for both oriPI-dependent and -independent transcriptional activation, indicating that the interactions are important for Cp activity. In transient transfections, we demonstrated that exogenous Oct-2 or Bright expression up-regulated oriPI-dependent Cp activation in the absence of EBNA1. Finally, endogenous Bright expression was shown to correlate with latency III but not latency I and II expression patterns in EBV positive cell lines, further supporting the notion that Bright expression is important for Cp transcriptional activity in vivo.