Epstein-Barr virus is a herpes virus that causes lymphoproliferative disorders in people and can immortalize human B-cells by infection in vitro. For one viral protein expressed in transformed cells, nuclear antigen EBNA1, at least one function is known. EBNA1 is required in trans for activity of the plasmid-maintenance element of EBV, oriP. One essential component of oriP, a family of 30-bp repeats, is activated in the presence of EBNA1 to become a strong transcriptional enhancer. The purpose of the proposed work is to determine how EBNA1 activates DNA replication and transcription and to evaluate its possible role in growth-transformation. A major goal is to purify sufficient quantities of EBNA1 from human cells to permit biochemical studies. Recombinant animal virus vectors, an infectious adenovirus vector or an EBV/SV40 plasmid vector, will be used to overproduce EBNA1 in cultured cells. This should allow the protein to be purified to near homogeneity in milligram quantities. The protein will be characterized biochemically, and efforts will be made to demonstrate its activation of replication and transcription in vitro. The eventual goal will be to determine with what cellular components EBNA1 interacts to stimulate these two activities. Mutations will be introduced throughout the EBNA1 gene in an attempt to separate the two activities and to complement the biochemical studies by defining the regions of the protein that are essential to its activities. Experiments will also be performed with plasmids in cultured cells to determine whether replication of oriP-carrying plasmids is limited by the amount of active EBNA1 or by a mechanism that limits DNA to one round of replication per cell cycle. A possible role for EBNA1 in growth-transformation will be evaluated by testing the capacity of the EBNA1 gene to alter the growth properties of nonestablished rodent and human cells in culture.
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