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The long-term goals of this research are to understand how the herpes simplex virus major DNA-binding protein, ICP8, moves into and within the cell nucleus, interacts with cellular and viral proteins and assembles into macromolecular structures to promote viral DNA replication and regulate viral gene expression. The specific aims are: (1) To define the mechanisms of intranuclear assembly of HSV DNA replication proteins by co-immunoprecipitation analysis of wt and mutant forms of ICP8 and other HSV DNA replication proteins to define the requirements for interactions of ICP8 with other viral proteins in infected cells and in vitro, by isolation of viruses expressing mutant ICP8 molecules to define the role of specific protein-protein interactions during assembly of prereplicative sites in infected cells, and by preparation and use of antibodies specific for individual portions of the ICP8 molecule to examine the accessibility of specific portions of ICP8 as replication structures assemble. (2) To determine the mechanism of the induction of the conformational change in ICP8 that occurs coincident with localization to prereplicative sites in the nucleus by defining the relationship between the conformational epitope and the DNA-binding function of ICP8, by providing biochemical evidence for a conformational change, by defining the minimal macromolecular requirements for the conformational change, by identification of host proteins that bind to specific forms of ICP8, and by mapping the conformational epitope recognized by the 39S monoclonal antibody. (3) To determine the role of ICP8 in the effects of HSV on the cell cycle by defining the function of the interaction between ICP8 and Rb by defining the sequences of ICP8 required for interaction with Rb, and isolation and characterization of a mutant virus defective for interaction of ICP8 with Rb, and by defining how HSV infection increases the level of p53 in infected cells. (4) To define the roles of ICP8 and other DNA replication proteins in regulation of viral gene expression by determining their effects on HSV gene expression from parental viral DNA templates. The integration of the results from these studies on protein assembly and regulation of gene expression should allow the formulation of a model of the mechanisms by which intranuclear localization and assembly of viral proteins and DNA affect the utilization of a DNA molecule for transcription and DNA replication. In addition, these studies should provide new understanding of nuclear replication events that could serve as targets for antiviral agents against the herpesviruses.

Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.