The long term goal of this research is to understand gene regulation in herpes simplex virus type 1. MOre specifically, the proposal is designed to explore the strategy used by the virus to autoregulate immediate early gene (which encode proteins important in the regulation of early/late genes) and to examine cis-acting sites in the immediate early promoters. Current tools of biochemistry, genetics and molecular biology will be used to examine two sequence specific DNA binding proteins: 1) ICP4 which is encoded by the virus, and is essential for virus replication, and; 2) a cellular DNA binding protein, which recognizes sequence motifs in viral immediate early enhancer/promoter elements. These virus and cell coded proteins will be purified to homogeneity and characterized with regard to their DNA binding activity and protein/protein interactions. The central theme of the proposal is to define at the DNA sequence level, protein contact sites (either by """"""""footprinting"""""""" experiments or other functional assays), mutate key residues in the binding domain to alter specific protein/DNA interaction and then introduce the mutation into cells to evaluate the response. Additional experiments are planned which address the importance of protein/protein interaction between ICP4 and ancillary transcription factors within selected viral promoter elements. The objective of the ICP4 experiments is to elucidate mechanisms by which ICP4 can activate some genes and repress others. The immediate objective of experiments with the cellular DNA binding protein is to determine the importance of this DNA binding protein in genetic activity and regulation immediate early promoter/enhancer regions. The work is significant and relevant to the viral infectious cycle because the immediate early genes are known to be essential in the lytic replication of the virus and represent a point in the life cycle of the virus where one can exert control over the outcome of the infection. Studies on the regulation of immediate early genes will also be useful in designing strategies to prevent reaction of the virus from a latent state.
