The long-term goals of this research are to define the mechanisms of action of the herpes simplex virus ICP8 ssDNA binding protein in viral DNA replication and late gene transcription and the mechanisms of assembly and maturation of viral replication compartments in the infected cell nucleus. In this application our specific aims are to: 1. Define the mechanisms of stimulation of late gene expression by ICP8 by: determining if ICP8 and ICP27 interact directly by studying in vitro expressed proteins, baculovirus expressed proteins, and fluorescence resonance energy transfer analysis, by mapping the portions of ICP8 that are needed for interaction with ICP27 and pol II holoenzyme, by determining if viral DNA synthesis and ICP8 bound to progeny viral DNA are needed continuously for late transcription, by performing chromatin immunoprecipitation studies to determine if ICP8 is needed to recruit Pol II to progeny viral DNA for late transcription. 2. Define the mechanisms of intranuclear movement of ICP8 and the mechanisms of formation of replication compartments by mapping the ICP8 sequences needed for movement to prereplicative sites and replication compartments in the infected cell nucleus, by defining the domains of ICP0 needed for promotion of replication compartment formation, by testing the need for metabolic energy and actin-based myosin activity for movement of ICP8 within the nucleus. 3. Determine the mechanisms by which the HSV UL31 and UL34 gene products promote late replication compartment maturation through the host chromatin and nuclear lamina by testing the hypothesis that UL31 and UL34 cause the phosphorylation of the lamin proteins, by testing the hypothesis that UL31 binds to lamins and ? causes their disaggregation, and by determining other proteins associated with UL31 and UL34 as candidates for effectors in nuclear structural changes. These studies are likely to provide insight into the mechanisms by which HSV assembles a new apparatus for viral DNA synthesis, late gene transcription, capsid assembly, and primary envelopment in the infected cell nucleus as well as potentially providing new targets for prevention and treatment of herpetic infections. ? ? ?
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