The long term goal of this study is to understand the effect of Epstein-Barr virus (EBV) infection on the biology of the infected B cell. 1) When EBV infects normal resting B cells it drives them to differentiate into proliferating lymphoblasts that grow indefinitely. One major goal of this study is to understand better the viral functions involved in activating the B cell with special emphasis on the role of circularization of the viral genome and expression of the EBV encoded membrane protein p63 (Encoded by the Eco R-1 Dhet fragment of EBV DNA). To perform this study we will measure levels of expression of latent viral cellular proteins (EBNA-1 EBNA-2 and p63) and cellular activation antigens (BLAST-1 and BLAST-2) in cells infected with intact viral DNA or irradiated DNA that cannot circularize. Relative levels of expression of the molecules will be assessed by immunofluorescence, Northern (RNA) and Western (protein) blots will also be performed for the viral proteins. The role of individual viral gene products will also be assessed by transfecting subgenomic fragments of EBV DNA into EVB- BL cells and measuring changes in cell surface phenotype with a battery of B cell specific monoclonal antibodies. 2) In vivo, the infected proliferating B cells are kept in check, in healthy individuals, by cell mediated immune (CMI) responses. The target structure (LYDMA) for CMI remains unknown. We will test various EBV encoded proteins (EBNA-1, EBNA-2, p63 and the virion glycoproteins gp 350/220) for their role at targets in CMI. Target cells that are autologous or HLA matched to a panel of EBV specific cytotoxic T cell line (CTL), will be transfected with appropriate fragments of the viral genome. Expression will be tested by Northern and Western blot analysis and immunofluorescence. Successfully transfected cells will then be tested for their susceptibility to lysis by EBV specific CTL. The possible role of the only EBV encoded membrane protein in transformed cells (p63) as LYDMA will also be tested by assessing the ability of various synthetic peptides, derived from the p63 sequence, to stimulate CTL. These studies should help us understand at the molecular level, mechanisms involved when EBV infects and activated B cells and how the healthy immune response eliminates the infected cells.
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