A characteristic common to DNA animal viruses is the expression early in infection of viral proteins which act in trans to regulate subsequent RNA polymerase II-dependent transcription of the remainder of the genome. Herpes simplex virus type 1 encodes several immediate early proteins which have been shown to affect the transcription of test genes in transient assays and also in the background of the viral genome. An understanding of how these transcriptional modifiers function to regulate viral and cellular gene expression is an important prerequisite to determine how HSV advantageously utilizes the host cell metabolic machinery during productive infection and how these mechanisms are altered resulting in latent infections in its human host. The predominant transcriptional regulatory protein specified by herpes simplex virus is the multifunctional immediate-early protein ICP4. ICP4 is required for the induced transcription of early genes, autoregulation of its own transcription and is also required for late events in the viral life-cycle. The studies proposed in this application have two long-term goals; 1) to determine the parts of the ICP4 protein which specify its know physical and biochemical properties. These properties include phosphorylation, intracellular localization, DNA binding, and multimerization. The activities of proteins will then be related to the relevant physical or biochemical properties. These studies will involve a genetic dissecting of the protein by the introduction and analysis of engineered viral mutants containing nonsense, deletion, and missense mutations in the ICP4 coding sequence, and 2) To determine how ICP4 regulates, both positively and negatively, viral gene expression. Particular attention will be given to the significance of ICP4 binding in the promoter domains of several specific HSV genes, and how these sites correlate with biological function. Lastly, the hypothesis formulated above will be directly tested in a fractionated in vitro transcription system to directly observe the effects of ICP4 and to determine the effect of ICP4 and viral infection in general on cellular transcription factors.
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