(as per abstract) The objective is to develop sufficient knowledge of the molecular mechanisms by which LMP1 alters cell growth and of the effects of inhibiting those mechanisms so as to design screens for inhibitors that might be effective in treating EBV associated malignancies. The specific goals are to: (1) Use recombinant EBV based genetics to clarify the role of specific LMP1 amino acids (aa) in primary B lymphocyte growth transformation. The focus will be on aa between transformation effector site 1 (TES1) and TES2, TES1, TES2 and aa within the transmembrane domains. (2) Determine whether LMP1 signals through interactions with other receptors. LMP1 association with membrane rafts, with cytoskeletal elements, and with other membrane proteins, particularly TNFRs, will be investigated using genetic, biochemical, and physiological approaches. (3) Determine the mechanisms by which N-terminally truncated IkB causes apoptosis of EBV transformed cells so as to better validate inhibition of NFkB as a therapeutic target. (4) Investigate the cytoplasmic effects of LMP1, including the molecular mechanisms by which LMP1 and CD40 recruit TRAFs, modify TRAFs, activate NIK and ASK, effect NIK and ASK1 interaction with downstream kinases, and continuously signal or return to a basal state. (5) Determine the effects of LMP1 on B lymphocytes and epithelial gene expression from the changes in RNAs following expression of LMP1, LMP1 TES1, LMP1 TES2, or LMP1 with null mutations in TES1 and TES2. The importance of NFkB, AP1, CREB, or other factor activations will be evaluated by analyses of relevant promoters, e.g., TRAF1, EGFR, and CD40. The effects of LMP1TES1 and LMP1TES2 on cell gene transcription and on transformation will be compared with the effects of LMP1 and LMP1/CD40 in transgenic mice. (6) Evaluate the role of TRAF3 in LMP1, CD40, and LTbetaR signaling through observing differences in RNAs with LMP1 expression or CD40 or LTbetaR activation in TRAF3 knockout versus normal mouse cells or mice. The role of PKN in TRAF3 effects on the cell cytoskeleton, membranes, or transcription will also be assessed. (7) Investigate the role of C. elegans TRAF3 homologue in C. elegans neural function so as to enable screens for interacting genes that might identify novel TRAF functions.
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