The purpose of this project is to continue the study of the EBV- encoded, growth-transformation associated nuclear (EBNA-1-6) and membrane (LMP) proteins. The mapping and definition of EBNA-4 and EBNA-6 will be continued. The work on EBNA-5 will be focused on the B95-8/Raji prototype negative lines and the possible existence of alternative specificities. We also intend to localize the EBNAs vs EA/VCA in the proliferating, compared to differentiating layers of the epithelium. The intranuclear distribution of the EBNAs will be studied at the EM level and compared to other nuclear proteins, such as c-myc, N-myc, cyclin and sn-RNPs. Proteins of the EBNA family will be affinity purified with the help of the existing monoclonals. The purified proteins will be tested for DNA dependent ATPase, and to poisomerase I and II activities. A modified McKay assay will be used to search for specific DNA binding sites on EBNA-2 and EBNA-5. EBNA-1, EBNA-2 and LMP transfected murine tumor lines will be tested for immunogenicity in syngeneic mice. Corresponding human transfectants will be tested for CTL- sensitivity in vitro, transformation associated changes and new activation markers. Phenotypic studies will be concerned with possible coordinated regulation of the EBNAs studied in sets of EBV-converted sublines, derived from the same EBV negative BL line. """"""""Mirror experiments"""""""" will be performed on myc-transfected LCLs that have shifted to a more BL-like phenotype. In order to extend the study of phenotypic regulation to other cell types, a C3d/EBV (CR2) receptor clones will be isolated from a cDNA library and expressed in various EBV negative cells. Receptor positive cells will be infected with EBV and the expression of the transformation vs. lytic cycle associated proteins will be followed. We shall also study the expression of all seven antigens in nasopharyngeal carcinoma biopsies.
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