Much remains to be learned about mechanisms cells utilize to defend against foreign DNA, both in the cytoplasm and nucleus, as well as how these defenses are subverted by viral countermeasures during infection. To address this knowledge gap, our studies of host defenses against foreign DNA focus on the poxvirus vaccinia and HSV1 as model pathogens. Understanding how these viruses accomplish DNA replication and transcription will yield new insights into the defense mechanisms cells use to respond to all foreign DNA. Toward this goal, our studies of vaccinia have led to the discovery that the poxviral B1 kinase is essential for viral DNA replication because it is needed to inactivate the cellular DNA binding protein BAF (barrier to autointegration factor). If it is not inactivated, BAF binds to viral DNA and acts as a defense against vaccinia DNA replication. Recent data from our lab suggests that BAF can act as an HSV1 inhibitor as well, indicating BAF may impair growth of multiple DNA viruses. BAF's antiviral activity likely occurs via its ability to compact and aggregate DNA to which it binds; we postulate that this compaction may contribute to host defense in multiple ways. For example, compaction likely limits the accessibility of the DNA to viral replication proteins via steric hindrance, and facilitates binding of other antiviral protein to the foreign genomes. Further examination of BAF's antiviral activity will yield unique insights int its mechanism of action. Our central hypothesis is that BAF initiates the assembly of DNA:protein complexes in a phosphorylation-regulated manner, providing a scaffold on which intrinsic defense effectors can converge. To test our hypothesis we propose three aims. AIM 1) Determine how phosphorylation and localization both regulate BAF's repression of poxviral DNA replication. These studies will yield insights into how post-translational regulation and localization provide interconnected mechanisms of regulating BAF's host defense activity. AIM 2) Determine how BAF- DNA complexes modulate transcription and DDR signaling to protect genomic integrity. Understanding how BAF and DNA repair machinery coordinate the silencing of foreign DNA, but are eluded by poxviruses, will be a central focus of this aim. AIM 3) Determine the mechanism whereby BAF acts as an antiviral against HSV-1 infection. Our data demonstrate that BAF can impair HSV-1 infection in a manner regulated by localization and/or phosphorylation, thus paralleling how BAF's anti-poxviral activity is modulated. These studies will yield insights into the molecular mechanism of BAF's activity against HSV-1. Throughout the course of these studies, we will compare and contrast how BAF works against both a cytoplasmic and a nuclear DNA virus, which will be an innovative application of these pathogens. As an outcome of this work we will better understand BAF's contribution to these nucleoprotein complexes and how BAF itself is regulated during viral infection. Thus, we will gain insight into cellular mechanisms which overlap in their ability to 1) silence foreign DNA through compaction and 2) protect genome integrity even in the absence of infection.