Our goal is to understand 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 B lymphocytes. The experimental approaches which have been employed to study other DNA tumor viruses have not generally been applicable to the study of EBV because: (i) the mRNAs encoding putative transforming functions are extremely low abundance; (ii) the viral transcripts studied to date are unusually complex; (iii) attempts to identity a region of the EBV genome responsible for growth transformation of human B lymphocytes have been unsuccessful, suggesting either that more than one viral antigen is required or that the transforming function(s) may be encoded in exons spliced from more than one region of the genome; and (iv) techniques used to generate other transformation-defective virus strains have not been applicable to EBV.
The specific aims are: (1) To clone as cDNAs and characterize the viral transcripts present in latently infected transformed human B lymphocytes including: (a) Identification of the 5' end of the transcript encoding EBNA-1; (b) Analysis of EBNA-2 and EBNA-3 and other related transcripts including mapping of their 5' termini; and (c) Characterization of the transcripts encoding EBNA's in the Burkitt's lymphoma cell line, P3HR-1, which produces nontransforming virus. (2) To study the expression of the three open reading frames recently identified within the transcript encoding EBNA-1 by: (a) Production of rabbit heteroantisera against Beta-galactosidase/EBV fusion protein products which will be used to screen latently infected cell lines for the expression of the putative viral antigens; (b) In vitro transcription and translation of a full length EBNA-1 cDNA. (3) To identify those viral functions essential for establishing and maintaining latency and growth transformation by site-directed insertional mutagenesis.
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