In order to mount intrinsic and innate immune responses to infections by DNA viruses, mammalian cells rely on specialized proteins that recognize the viral DNA as a foreign molecule. Upon binding to viral DNA, these sensors induce cytokine secretion, prompting neighboring cells to activate their defenses and inhibiting the spread of infection. Recent years have seen significant progress in the understanding of processes governing viral DNA sensing. Contrary to prior dogma, we determined that mammalian cells can distinguish viral DNA from self-DNA in the nuclei of infected cells. The characterization of the interferon inducible protein IFI16 as the first known nuclear sensor of viral DNA has opened a new research direction in immunity, starting to shed light on how cells detect nuclear-replicating viruses, such as herpesviruses. We further uncovered that the human pathogens, herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) have immune evasion mechanisms that specifically suppress IFI16. With this knowledge, during the previously funded R01, we aimed to dissect the mechanisms underlying nuclear IFI16 DNA sensing. We addressed questions regarding where and when the IFI16-viral DNA binding event occurs, the properties that allow IFI16 to sense DNA, and the functional interactions that support IFI16 antiviral responses. Our results uncovered that nuclear DNA sensing relies on dynamic on/off sensor associations with parental viral DNA at the nuclear periphery. We demonstrated that IFI16 oligomerization on viral DNA is essential for nucleus-derived immune signaling. We further established that, upon binding to viral DNA, IFI16 triggers both cytokine expression and suppression of viral gene expression. Our proposal will address several fundamental questions regarding nuclear DNA sensing that have emerged from these findings. In Aim 1, we will define what mechanisms drive dynamic IFI16 oligomerization with HSV-1 DNA at the nuclear periphery. We will test our hypothesis that this property is biophysically conferred via rapid and reversible liquid-phase condensation events, and delineate how these events govern innate immunity and viral replication. Next, we will characterize mechanisms underlying the two antiviral IFI16 functions downstream of its binding to DNA. In Aim 2, we will determine how IFI16 induces innate immune signals upon nuclear DNA sensing. We discovered that IFI16 interacts with and activates the DNA dependent protein kinase (DNA-PK) holoenzyme in response to herpesvirus infections. We will define how IFI16 and the DNA damage response coordinate to stimulate innate immunity and antiviral non- homologous end-joining. In Aim 3, we will elucidate the mechanisms underlying IFI16 restriction of virus gene expression. We will functionally characterize the IFI16 interactions with chromatin modulators that we showed to act as HSV-1 restriction factors. We will systematically define the pathways linking IFI16 to viral genome hete...