The long-range goals of this research continue to be the elucidation of the mechanisms by which Epstein-Barr virus (a DNA virus associated with several human neoplasms) establishes and maintains latency and growth transformation of infected human Beta lymphocytes. Furthermore, regulation of EBV gene expression affords a moderately complex system to study the general mechanisms of control of eucaryotic gene expression. The focus of this application is on continuing to characterize the viral transcripts present in latently infected cells and any associated open reading frames. In addition, control of latent gene expression will be a major new focus of this competing renewal. The scope of this application will be limited to the characterization of rightward viral transcription of those messages containing 5' exons from the major internal repeat, IRl. These goals will be approached as follows: 1. Characterization of viral transcripts in latently infected lymphocytes, by: (a) preparing and screening cDNA libraries from appropriate Burkitt's lymphoma and lymphoblastoid cell lines; (b) primer extension, Sl nuclease protection and nuclear run-on assays to identify transcription start sites and their associated promoter regions; and (c) field-inversion electrophoretic gel analyses of viral DNA to determine the size of the major internal repeat, IRl. 2. Analysis of the cis and trans-acting elements involved in the control of viral transcription in latently infected lymphocytes, by: (a) employing CAT assays to determine the influence of upstream and downstream sequences on the activities of the IRl and Ul encoded promoter elements, in EBV-positive and negative Beta lymphocytes; and (b) DNAse I footprinting and purification of DNA-binding factors involved in regulating viral transcription. 3. Identification of viral antigens associated with latent infection, by: (a) immunizing rabbits, with either bacterial fusion proteins or synthetic peptides coupled to a carrier protein, to generate monospecific heterosera against the putative viral antigens; and (b) immunoblotting, immunofluorescence and cellular fractionation employing the rabbit antisera to characterize the expression and cellular distribution of the putative antigen.
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