Infection of human B cells by Epstein-Barr virus (EBV) leads to unrestricted cell growth and transformation in vitro under conditions of immunosuppression. Although the precise molecular interactions which mediate the initial contact of EBV with its cellular receptor (CR2) have been elucidated, comparatively little is known of how the virus subsequently penetrates the barrier of the host cell plasma membrane. This proposal will carry out a comprehensive analysis of EBV entry into B lymphocytes and epithelial cells at the molecular level. The nature of the interaction of the EBV envelope glycoprotein, gp85 with host cell membranes including binding to a cellular receptor will be examined. Physicochemical and ligand binding studies will be used to determine whether other EBV envelope glycoproteins such as gp350/220 interact with gp85 following CR2 binding and thereby induce conformational changes in gp85 which then influences virus entry. Further studies will identify the EBV protein(s) which form a complex with gp85, including the HSV-1 envelope glycoprotein gL homologue. These studies will determine whether gp85-associated proteins regulate posttranslational modification, transport and proper folding of the gp85 protein. A direct role of gp85-associated proteins in virus entry into cells will also be ascertained. Finally, we will induce human antibody responses to EBV gp350/220 and gp85 in SCID/human mice as a means to determine the structure of the EBV proteins and to analyze the role of viral replication during the development of EBV-induced lymphoproliferative disease. Molecular biological approaches will be used to map human neutralizing Fab binding sites on gp350/220 and gp85. Large single crystals of soluble gp85 or of co-crystals of gp85 and neutralizing Fab will be grown in preparation for solving the three-dimensional structure of gp85. These studies will provide fundamental knowledge of the molecular mechanisms of EBV entry into host cells and may facilitate the development of additional strategies for the intervention of EBV-induced disease in man
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