The differential stability of interactions between kinetic classes of HSV promoters and the basal transcriptional machinery of the cell combined with virus-mediated compartmentalization of transcription machinery does much to explain the selective expression and repression of various kinetic classes of viral genes during the different phases of the productive replication cycle. We will expand and validate this model by accomplishing the following: 1. Analyze the biochemical interaction between a class of strict late promoters that contain a downstream activating sequence (DAS), as exemplified by the UL38 promoter, and the DNA binding subunits (Ku) of the multifunctional cellular enzyme DNA-dependant phosphokinase (DNA-PK). A major feature of this investigation will be the use of cultured cells in which components of DNA-PK have been functionally deleted, and purified TFIID. 2. Choose model promoters to investigate other modes that HSV utilizes to directly stabilize the interaction between late promoters and the TFIID complex. HSV DNA micro-arrays will be developed for this study. 3. Investigate how the kinetic class-specific promoter structure of HSV transcripts influences the strength of binding of the pre-initiation complex to influence time of maximal expression. We will use purified TFIID for biochemical studies, as well as in situ hybridization methods for analysis of differential gene expression in individual cells. 4. Use cell culture and mouse pathogenesis models to study of the precise role of time and level of maximal expression of selected required viral genes. This work has been started with viruses expressing kinetic alterations in the major capsid protein (VP5). We will also study kinetic modifications of expression of the VP19 capsid protein, the virion trans- activating protein, and the immediate-early ICP 27 promoter.
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