Epstein-Barr virus (EBV) is associated with the development of several malignancies including Burkitt's Lymphoma, nasopharyngeal carcinoma, and B cell lymphomas in immunocompromised individuals. The viral proteins expressed during latent, growth transforming EBV infection of B lymphocytes have been identified. The role of some EBV proteins in latent, growth transforming infection, namely EBNA-1, EBNA-2, and LMP, have been partially delineated. However, except for EBNA-2, the latent viral genes required for growth transformation have not been identified. Furthermore, there is little known about EBNA-LP, EBNA-3A, EBNA-3B, EBNA-3C and terminal protein function. The goal of this proposal is to undertake a genetic analysis of the latent EBV genes involved in growth transformation. Recombinant EBV genomes will be used to identify the latent viral genes required for EBV induced growth transformation and allow study of specific gene function during virus infection and B lymphocyte growth transformation. Specific recombinant EBV will be generated by using 1) naturally occurring EBV deletion mutants to reconstitute transformation with natural or mutated genes or 2) homologous recombination of a positive selection marker and selection in either EBV negative B lymphoma cells or B lymphocytes. Specifically, the aims will be as follows: 1) EBNA-LP function and requirement in growth transformation will be studied using P3HR-1 virus recombinants. 2) EBNA-3C, EBNA-3A and EBNA-3B, function and role in growth transformation will be studied by recombination and restoration of the Raji EBNA-3C and BALF2 deletion. 3) LMP function and its role in growth transformation will be studied insertional mutagenesis by homologous recombination of a drug selectable marker. EBV genetic analysis can identify the viral genes required for growth transformation and allow study of their function directly in EBV induced B lymphocyte growth transformation. This is a necessary corollary of ongoing studies of individual EBV gene effects in order to better understand EBV infection and its etiological relationship with human malignancies.
