The Herpes viruses are large DNA viruses several of which infect human cells to cause a myriad of diseases some, severe. Combating these viruses will require knowing more about their life cycles. The initiation of DNA replication presents an attractive target for anti-viral approaches as it represents the earliest stage in the production of new virus. Herpes Simplex type l (HSV- l) infects human cells and is the best understood of the Herpes viruses. In this project, efforts will continue to focus on the action of viral and host proteins in the first stages of initiation of HSV- l replication, in particular UL9 protein which binds to the HSV-l origins and acts as a helicase, and ICP8, the general single strand DNA binding protein. A major focus will be on further characterizing the interactions of ICP8 and UL9 as they open and unwind the HSV-l origin. Analysis combining biochemical and electron microscopic (EM) studies will provide a detailed mechanism including how topoisomerase I drives the unwinding reaction and whether it binds directly to UL9. It is possible that host cell heat shock (chaperone) proteins help load UL9 onto the origins and this possibility will be explored. These studies will employ EM, biochemical assays, and surface plasmon resonance measurements to define the nature and structure of the protein complexes that form at the origins and initiate replication. Using plasmid DNA-protein complexes in which the origin is partially unwound by UL9 and ICP8, extracts from HSV-1 infected human cells and an HSV-l replisome generated in insect cells will be added to learn more about the subsequent steps of replication. The long-range goal is to reconstitute full HSV- l replication using plasmid templates in vitro. Activation of latent HSV- l and initiation of replication from latent genomes likely requires removal of nucleosomes from the origin. A GRE (glucocorticoid response element) has recently been identified in the HSV-l origin termed oriL and this may act to uniquely position nucleosomes over the UL9 binding sites in oriL, creating a molecular, hormone-sensitive switch. This will be tested using in vitro chromatin assembly and footprinting methods. High resolution EM will be utilized to determine the fine structure of several HSV- l DNA-protein complexes. A collaborative project with Dr. Kenney of this program project will explore many of these same questions in the EBV system.