Pseudorabies virus infected cells will be used as a model system for studying the mechanism of activation of eukaryotic gene transcription. The ICP4 protein of this virus regulates the transcription of early herpesvirus genes and induces the transcription of unrelated adenovirus genes in coinfections. In addition, ICP4 activates unrelated cell genes in cotransfection assays. Thus ICP4, as well as the adenovirus E1A protein, may be activating transcription indirectly, rather than by binding to specific DNA sequences as prokaryotic regulatory proteins do. ICP4 will be purified by monoclonal antibody affinity chromatography and its biochemical properties analyzed for the purpose of understanding transcriptional activation. Monoclonal antibodies to ICP4 will be used to search for interactions between ICP4 and transcriptional regulatory proteins of the cell. Since the ICP4 protein is 180K and possibly multifunctional, deletion mutants within ICP4 will be made and recombined into virus to correlate the loss of biochemical properties of ICP4 with the loss of the transcriptional activation function. Finally, we will continue our investigations into the activities of the temperature sensitive ICP4 protein by determining whether the expression of genes present in different states can be inhibited by this protein. A gene with a very strong enhancer sequence will be transfected into cells and infected with tsG at the nonpermissive temperature to determine if strongly enhanced genes are inhibited. A series of cell lines containing integrated adenovirus early genes will be tested to determine if the same genes present in chromatin can be repressed by tsG infection. Our knowledge of the genes which are activated and the ability to purify the protein responsible for their activation gives us an excellent system for studying transcriptional activation in eukaryotic cells.
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