FUCTIONS OF EBV PROTEINS IN B CELL IMMORTALIZATION

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The long term objective of this research is to discern the mechanism by which Epstein-Barr virus induces proliferation and immortalization of B cells; EBV-induced proliferation can lead to lymphomas in immunosuppressed individuals. This information is essential to our understanding of the pathogenesis and persistence of EBV as well as our comprehension of the process of B cell proliferation, a highly regulated event central to immune function. Latent infection by EBV results in expression of 6 viral nuclear proteins (EBNAs) and 3 viral membrane proteins followed by proliferation and immortalization of the infected B cells. We propose to identify the functions of the latent infection proteins and the cellular pathways through which they act. Specifically, we propose to: 1) determine a biochemical basis of EBNA2-induced immortalization of B cells, 2) elucidate EBNA function by characterization of associated cellular proteins and 3) establish the biochemical effects of individual EBV latent infection proteins in primary B cells. Our preliminary studies have indicated that interaction of one of these nuclear proteins, EBNA2, binds to the retinoblastoma protein, Rb, and that EBNA2 contains sequences which are homologous to the Rb-binding domain of SV40 large T antigen. We propose to determine whether the interaction is mediated via these sequences by using specific peptides to inhibit the interaction and by examining the ability of EBNA2 deletion mutants to bind Rb. The EBNA2 sequence differs in some residues which are believed to be invariant and therefore these results will provide information about the relative importance of these residues. We have recently demonstrated that other cellular proteins bind to the EBNAs; these cellular proteins are likely to include not only proteins which have been demonstrated to associate with DNA tumor virus transforming proteins, such as Rb, but also proteins which are involved in the exquisite control of B cell proliferation as well as proteins involved in other cellular processes such as transcription factors. Using EBNA fusion proteins to screen a cDNA expression library, we propose to isolate the cDNAs encoding cellular proteins which bind to the EBNAs. The role of individual viral proteins will be investigated by transfection of viral genes into primary B cells; we will examine activation markers and radiolabelled nucleotide incorporation to determine the effects on activation and proliferation of B cells. These studies will allow us to define the biochemical function of the EBV latent proteins.