Year: 2008
Type: dissertation
Abstract: Epstein-Barr Virus (EBV) is an exclusively human, lymphotropic herpes virus\nthat infects more than 90% of the population worldwide. Primary infection\nusually occurs during the early years of life and does not result in any\nrecognized disease. EBV is the causative agent of infectious mononucleosis and\nis associated with various malignancies including Burkitt’s lymphoma (BL),\nHodgkin’s disease (HD), nasopharyngeal carcinoma (NPC), and immunoblastic\nlymphomas in immunocompromised individuals. In most immunocompetent\nindividuals the virus is, however, harbored for life within latently infected\nresting memory B cells, causing no symptoms. In vitro, EBV efficiently\ntransforms resting B cells to activated lymphoblasts. These perpetually dividing\ncells express a repertoire of viral antigens (EBNA1-6 and LMP1), all of which\nhave been directly implicated in the immortalization process. Immediately postinfection,\nEBNA2 and -5 are the expressed from the W promoter (Wp), within\n36 hours there is a switch in promoter usage from Wp to the upstream\nC promoter (Cp). Transcription from Cp leads to a concomitant expression of all\nEBNAs from a polycistronic transcription unit that is spliced to yield the\ndifferent EBNA proteins. EBNA1 forms multiple homodimers that bind to a\nportion of the latency origin of replication (oriPI) that functions as an EBNA1-\ndependent enhancer of the Cp. The mechanism for the interaction between the\noriPI-EBNA1 complex and the Cp is not completely understood at the\nmolecular level. EBNA1 has no apparent enzymatic activities and is thought to\nfulfill its functions by mediating interactions with specific host cellular proteins,\nonly few of which have been characterized. The aim of this thesis was to\nidentify and characterize the interaction partners of this macromolecular\ncomplex.\nThe interactions of the transcription factors NF-Y and Sp1 with the promoterproximal\nregion of the Cp were previously established in our lab. In paper I we\nstudied these interactions further using transient transfections, establishing that\nNF-Y and Sp1 co-stimulate Cp and that the oriPI-EBNA1-induced\ntransactivation of Cp requires concomitant expression of both proteins.\nFurthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2\nfunction is regulated by estrogen, we demonstrated that inactivation of EBNA2\nresulted in decreased expression of NF-Y and down-regulation of Cp. Knowing\nthat resting B cells do not express NF-Y and observing that this factor is\nessential for Cp activation, we suggest that its up-regulation post-infection may\ncontribute to the Wp-to-Cp switch in primary EBV infection. The oriPI contains\n20 repeats of the EBNA1 binding domain. In paper II we used a series of oriPIdeletions\nin oriPI-CpCAT reporter plasmids in transient transfections to\ndetermine the number of EBNA1 binding repeats necessary for efficient\ntransactivation of the Cp. We showed that eight or more repeats were necessary\nfor this effect, which underscores the complexity of the transactivation process.\nIn papers III and IV we set out to identify novel interaction partners of the oriPIand\n-170Cp regions using EMSA and DNA affinity purification coupled with\nmass spectrometry. Three novel protein interactions with the oriPI and the Cp\nwere identified. The transcription factors Bright, E2F1 and Oct-2 were found to\nbind both sequences in vitro and in vivo, opening up a possibility of mediating a\nlink between the oriPI and the Cp. The binding sites of all three proteins were\nmapped to a short segment of Cp in close proximity of each other. This region\nwas previously shown to be essential for both oriPI-dependent and -independent\ntranscriptional activation, indicating that the interactions are important for Cp\nactivity. In transient transfections, we demonstrated that exogenous Oct-2 or\nBright expression up-regulated oriPI-dependent Cp activation in the absence of\nEBNA1. Finally, endogenous Bright expression was shown to correlate with\nlatency III but not latency I and II expression patterns in EBV positive cell lines,\nfurther supporting the notion that Bright expression is important for Cp\ntranscriptional activity in vivo.
Author: Cecilia Boreström
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W655599964