INITIATION OF CELL INFECTION WITH EPSTEIN-BAR VIRUS

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Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that causes infectious mononucleosis, is associated with Burkitt's lymphoma and has been implicated in the etiology of nasopharyngeal carcinoma. It contributes to the development of lymphoproliferative syndromes and is thought to influence the pathogenesis of the human immunodeficiency virus. Recruitment of normal cells by endogenous virus, produced in increased amounts during episodes of immunosuppression, is of great relevance to virus induced pathology and probably contributes to development of the premalignant state. The overall objective of this proposal is to understand how EBV enters its two targets, the B lymphocyte and the epithelial cell and is based on the premise that clarification of how this process occurs is critical to rational design of chemical and biologic inhibitors of disease. Work in the previous award period implicated one of the virus envelope glycoproteins, gp85, in fusion of EBV with the lymphocyte membrane and provided evidence for additional novel proteins that may contribute to early events in infection. The goals of the current proposal are to continue study of virus entry and the envelope proteins involved in the process, to delineate functional domains of glycoprotein gp85, and to test the hypothesis that gp85 is a virus fusion protein. There are three specific aims. The first is to determine the function and structure/function relationships of gp85. A panel of monoclonal antibodies will be mapped to different regions of the protein and their ability to neutralize and block virus penetration will be used to identify regions that are of particular functional importance. A cDNA clone of gp85 will be expressed in vaccinia virus to make enough protein for the antibody mapping and to establish the orientation and anchor sequence(s) of the molecule. Virosomes will be used to make artificial "deletion mutants" of gp85 and an effort will be made to obtain quantities of gp85 sufficient to analyze its functions after insertion into liposomes. The second aim is to characterize biochemically and functionally the three novel EBV-induced membrane proteins. One protein will be sequenced and antibodies will be made to synthetic peptides derived from candidate open reading frames to map the other two to the viral genome. the third aim is to use a fusion assay for EBV to examine parameters that influence internalization and to determine the relative importance of different envelope proteins to entry into B cells and epithelial cells.