Herpes Simplex Virus (HSV) is a ubiquitous human pathogen that can cause significant morbidity and mortality in humans. The survival of all organisms including viruses depends on their ability to produce an exact copy of their genetic material. The DNA replication machinery (replisome) functions as a complex association of proteins that must be tightly regulated in order to accomplish faithful and complete DNA replication. Our previous work has led to the identification and characterization of seven HSV replication proteins as well as the compartments in the cell in which DNA replication occurs; however, the mechanisms by which they function during DNA replication are poorly understood. Recent experiments by us and our collaborators suggest that the assembly and function of the HSV-1 replisome is tightly regulated by protein-protein and protein-nucleic acid interactions that we are only now beginning to appreciate. Our central hypothesis is that protein-protein interactions among viral replication proteins are essential for all stages of genome replication. We will examine the roles of specific interactions in the formation of prereplicative sites and replication compartments, initiation of DNA synthesis at viral origins of replication, processive unwinding of the duplex DNA in front of the replication fork and the coordinated regulation of leading and lagging strand DNA synthesis. HSV dramatically reorganizes the infected cell nucleus leading to the formation of large globular replication compartments in which gene expression, DNA replication and encapsidation occur. Although it has long been recognized that ICP8 is required, little is known about how ICP8 exerts its effects on the nuclear architecture. In aim 1 we will use a combination of genetic, biochemical, biophysical and cell biological methods to test the hypothesis that dynamic properties of ICP8 and its ability to assemble into small subassemblies and filaments drive the formation of prereplicative sites and replication compartments. ICP8 and the origin binding protein UL9 are known to work together to unwind the origins during the initiation of DNA synthesis; however, difficulties in reconstituting origin dependent DNA synthesis in vitro have prevented a thorough understanding of the process. In aim 2 we will test the hypothesis that protein-protein and protein-DNA Interactions are essential for origin dependent initiation of viral DNA synthesis. We will use genetic and biochemical approaches to identify the regions of UL9 and ICP8 that are important for various steps in origin unwinding. In aim 3 we use a newly developed assay for coordinated leading and lagging strand synthesis to test the hypothesis that interactions between the HSV helicase/primase and polymerase are required for coordinated leading and lagging strand DNA synthesis.