In this application, Dr. Speck describes approaches for extending his laboratory's analysis of viral transformed human B cells. The long range goals of this research continue to be the elucidation of the mechanisms by which EBV regulates viral gene expression in latently infected, growth transformed lymphoblastoid cell lines. The scope of this application is limited to the characterization of the complex transcriptional unit which spans the major internal repeat of the virus (IR1) and gives rise to the various viral EBNA mRNAs. This transcriptional unit spans ca. 100 kb of the viral genome and is under the control of two promoters, Cp and Wp, located near the left end of the linear viral genome. The applicant proposes an evaluation of a promoter-switching model for the early stages of EBV infection of B lymphocytes. According to this model, during the initial stages of viral infection, Wp is used exclusively to drive transcription of the genes encoding EBNAs 2 and 4, with a subsequent switch to Cp usage and expression of all six EBNA genes. Since an EBNA2-dependent enhancer has been identified in the region upstream of Cp, it is also proposed that EBNA2 plays an essential role in driving the Wp to Cp promoter switch in primary B lymphocytes. To investigate this model the applicant proposes to: (1) identify and characterize the critical cis-elements involved in mediating mutually exclusive Cp and Wp usage; (2) characterize lymphoblastic cell lines that exhibit constitutive Wp activity; (3) analyze viral promoter switching during the initial stages of infection; and (4) characterize the cellular factors interacting with the Cp associated EBNA2-dependent enhancer.
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