Epstein-Barr Virus (EBV) is a human pathogen that is causally associated with both benign and malignant B-cell lymphoproliferations and nasopharyngeal carcinoma. Immunocompromised individuals such as AIDS patients and transplant recipients are at high risk for developing EBV- associated B-cell lymphomas. Virally-induced alterations of the growth properties of infected B-cells are likely to underlie EBV's contribution to these lymphoproliferative disorders. Infection of primary human B- cells in vitro EBV results in activation of the resting B-cell from a quiescent state to one of proliferation. These infected cells enter the cell cycle and are able to proliferate indefinitely and thus are immortalized. EBV is unique in its ability to immortalize primary resting B-cells in vitro. Entry of EBV-infected B-cells into the cell cycle and maintenance of the proliferative state of the cell is absolutely dependent upon viral gene expression. The overall aim of this proposal is to analyze the contribution of one particular gene product (LMP-1) of EBV to immortalization. The study of LMP-1 is of particular significance for the following reasons: 1) LMP-1 is one of 4 latent gene products, and the only membrane protein, shown to be essential for immortalization by EBV; ii) LMP-1 has profound effects on the growth of cells in culture (i.e., LMP-1 functions as an oncogene in rodent cells); iii) LMP-1 is located in the plasma membrane where it is associated, via its cytoplasmic amino- terminus, with the cytoskeleton. LMP-1 must interact with cell signal transduction pathways regulating normal B-cell proliferation and thus is the ideal starting point for identifying signaling pathways involved in immortalization by EBV. The specific objectives of this work are to use biochemical and genetic analyses of LMP-1 to: i) determine the transmembrane topology of LMP-1; ii) study the quaternary structure of LMP-1 and its relevance to function; and iii) determine the relationship between the structure of LMP-1's amino- and carboxy-termini and function, cytoskeletal association, turnover and patching. These experiments are expected to provide information that will aid in the identification of LMP-1's biochemical activity that results in altered cellular phenotypes and should allow the generation of rational models of how LMP-1 may function in EVB-mediated immortalization. Understanding the signal transduction mechanisms resulting B-cell immortalization by EBV will provide insights into the cellular mechanisms controlling the normal and malignant proliferative status of human B-cells, and may provide the basis for clinically relevant research aimed at the development of therapies for the prevention and treatment of EBV-dependent lymphomas as well as other virally and nonvirally induced malignancies.
