Epstein-Barr Virus (EBV) causes infectious mononucleosis in adolescents and malignant B lymphocyte proliferation in immune compromise patients, in marmosets, or upon transfer of infected human B lymphocytes into SCID mice. EBV is also etiologically associated with African Burkitt's lymphoma and nasopharyngeal cancer. EBV transformed, latently infected B lymphocytes contain EBV episomes and eight virus encoded proteins. Six are nuclear proteins (EBNAs) and two are the integral membrane proteins, LMP1 and LMP2. These eight proteins are presumed to mediated latent virus infection or B lymphocyte proliferation and are thus under intense scrutiny. Beside EBNA1, which is required for episome maintenance, LMP1 and LMP2, are the two transformation associated proteins that are most consistently detected in EBV related malignancies, and the LMP2 message is the only message detected in PCR analysis of B lymphocytes from individuals harboring EBV latent infections. These observations argue for an important role for LMP2 in the biology of EBV. LMP2 might may control activation of lytic replication or down regulate the activation state of EBV infected cells allowing persistence in the human host. The elucidation of LMP2 function in latent virus infection, B lymphocyte proliferation, or maintenance in the human host utilizing biochemical and genetic techniques will be focus of this research proposal. An understanding of LMP2's function may provide insight for eh development of novel therapeutics for the treatment of ENV related malignancies. The following aims are proposed: 1) Identify LMP2 associated proteins. LMP2 associates with LMP1, src family tyrosine kinases, and at least three unidentified cell proteins, one of which is tyrosine phosphorylated. 2) Introduce site specific mutations into LMP2 and src to delineate amino acids critical for the protein-protein interactions between src and LMP2 and other cell proteins (Aim 1). 3) Investigate the phenotype of non EBV infected B lymphoma cells expressing LMP2. Cell lines expressing LMP2 are unable to signal through surface receptors. LMP2 mutants from aim 3 will be tested for signalling defects. 4) Characterize EBV immortalized cell lines which are transformed with EBV recombinants fully null for LMP2 expression. Characterization will include analysis of cell surface marker expression, in vivo growth properties in SCID mice, and activation of lytic infection following crosslinking of cell surface receptors.
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