REGULATION OF REPLICATION AND LATENCY BY EBV EBNA-1

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Epstein-Barr Virus (EBV) causes infectious mononucleosis and is carried as a latent infection by 80% of the adult American population. Individuals who are immunologically compromised, such as AIDS victims, and patient undergoing organ or bone-marrow transplantation, frequently develop symptoms of re-activated EBV infection. Incidences of EBV-positive lymphomas in these patients has also been a cause for concern. An understanding of the factors involved in viral replication and maintance of latency would, in the long term, assist in the management of these patients. EBV is also believed to be a causative factor in Africa Burkitt lymphoma and nasopharyngeal carcinoma (which occurs pedominatly in Chinese ethnic groups) since the cells of these tumors contain EBV DNA and express the EBNA-1 antigen. The long range goals fo the proposed research is to define the molecular mechanisms involved in the replication of Epstein-Barr Virus DNA and in the maintenance of viral latency in transformed cells. The specific aims of this application are to investigate the biological and biochemical processes involving the EBV nuclear antigen EBNA-1 and the regions of EBV DNA with which it interacts and will include: 1. Isolation of the intact EBNA-1 protein. 2. Examination of the parameters of binding of EBNA-1 to its DNA binding sites. 3. Examination of the biological implications of manipulating the numbers and affinities of the EBNA-1 binding sites in ori-P. 4. Determination of the minimal region of EBNA-1 required for specific DNA binding, plasmid maintenance, nuclear localization of EBNA-1, and chromosomal association of EBNA-1. 5. Invesitgation of a possible role for EBNA-1 in regulation of biral gene expression, and 6. Identification of lytic EBV orgins of replication. Specific reagents will be generated using recombinant Dna thechnology and assay procedures will include DNAaseI footprinting, nitrocellulose filterbinding assays, DNA transfection, CAT assays and immunoflorescene.