Epstein-Barr Virus (EBV) is a human herpes virus which infects and transforms B lymphocytes. It is the causative agent of infectious mononucleosis and is strongly associated with two human tumors, Burkitt's lymphoma and nasopharyngeal carcinoma. Details of the molecular biology of this virus are still poorly understood; such studies have been severely hampered by the lack of an in vitro system which is completely permissive for viral replication. Although the entire DNA sequence of EBV was recently determined, very few gene products have been assigned to specific open reading frames. We propose to identify and map the genes for EBV nucleocapsid components and to initiate studies on the regulation of EBV gene expression by studying the control of EA (early antigen) synthesis. We have developed a method for the purification of biologically active EB virions. We now propose to use these virions to develop monoclonal antibodies specific for individual virion components; these antibodies will be used to screen for expression of the protein in cells transfected with various regions of the EBV genome inserted into expression vectors. Once the general region coding for a particular component has been identified, the precise boundaries of the gene will be delineated by S1 nuclease mapping. The results of these experiments will identify the various polypeptides which comprise the EB nucleocapsid and determine which segment of the EBV genome codes for them. In addition, as the first step in understanding the control of viral replication in B lymphocytes, we will investigate the mechanism by which the synthesis of early antigen is controlled. EA appears to be one of the first gene products expressed at the start of the replication cycle. The putative genes for EA will be placed in an SV40 expression vector to verify that these genes do, in fact, code for the EAs previously identified in EBV positive cells using specific antibodies. The upstream regulatory sequences of these EAs will be studied for their role in the chemically induced expression of EA using site-specific mutagenesis. The results of these experiments will greatly enhance our knowledge of EBV replication and of the control of EBV gene expression. The information gained concerning the regulation of EA expression is likely to reflect a general mechanism of eukaryotic gene control.
