EBV is a ubiquitous human pathogen which persists in infected individuals for life in latently infected B lymphocytes. The major goal of the proposed research is to understand the mechanisms involved in controlling the Epstein-Barr Virus (EBV) BZLF1 gene promoter (Zp), which is integrally involved in the switch of the virus from latent infection to the viral lytic life cycle. It is important to understand how this switch is kept """"""""off"""""""" during viral latency, and what the physiological signals are for activation of the viral lytic life cycle. An initial characterization of Zp has been carried out, and has revealed two regions involved in TPA inducibility and a third distinct region involved in transactivation of Zp by the product of the BZLF1 gene. Further characterization of control of Zp activity will involve the following: (1) Further characterization of the functional domains in Zp; (a) Identification of binding domains in Zp for nuclear factors employing affinity purified extracts; (b) Mutational analysis of ZI domains; (c) Cloning of Zp domains upstream of heterologous promoters; (d) Characterization of 3' deletions and random scanning mutations; (e) Analysis of activity and DNA-protein interactions in non-B cells; (f) Effect of theophylline on promoter activity in the Ramos cell line; (2) Purification and characterization of transcription factors interacting with Zp; (a) Preliminary characterization of factors interacting with Zp by gel retardation, UV crosslinking, microscale DNA affinity precipitation, and in vivo footprinting; (b) Purification and characterization of transcription factors interacting with Zp by affinity chromatography and by oligonucleotide screening cDNA expression libraries; (3) Analysis of the time course of BZLF1 gene expression as a function of cell line and lytic cycle inducer; (4) Investigation of the role of EBV antigens in modulating Zp activity; (a) Possible role of EBV gene products expressed during latency in modulating Zp; (b) Role of viral antigens expressed during the early phase of the lytic cycle in modulating Zp activity; (5) Characterization of the effect of B cell activation and differentiation on the induction of the viral lytic cycle and the activity of Zp.
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