ABSTRACT The human herpesviruses are responsible for lifelong debilitating and congenital infections, and some members of this family are associated with human cancers. HSV causes significant disease during acute infection and establishes persistent latent infections in sensory neurons for the life of the host leading to reactivation and recurrent disease. This proposal is based on a series of observations about the interplay between HSV infection and the intrinsically antiviral cellular protein, PML. In response to interferon treatment, oxidative stress, DNA damage, and viral infection PML oligomerizes to form a mesh-like spherical shell around heterogeneous, phase- separated nuclear condensates, called PML nuclear bodies (PML-NBs). PML oligomerization leads to the activation of its SUMO ligase activity, which in turn induces the recruitment of dozens of cellular proteins that contain SUMO-interacting motifs (SIMs) including repressors of gene expression into PML-NBs via SUMO/SIM interactions. PML-NBs are anti-viral, however, the exact mechanism of their action remains poorly understood. PML-NBs size and number increase in response to HSV infection, and they are actively targeted for degradation by HSV1 as a mechanism to counteract their antiviral effects. During HSV infection PML-NBs have often been observed adjacent to the sites of viral DNA replication and have recently been shown to entrap the entire viral genome. Despite a rudimentary understanding of these processes, many questions remain about how PML-NB formation is triggered by HSV infection and how they are recruited to viral genomes. Oligomerization and sumoylation of PML have been shown to be essential for the effective formation of PML-NBs in response to stress. In Aim 1 we will identify PML domains that are necessary for its SUMO-E3 ligase activity and required for formation of the PML-NBs. One of the most surprising aspects of PML-NB formation is their ability to form around and entrap viral DNA, resulting in genome silencing. The properties of PML that promote this unusual ability to recognize viral DNA are not understood. In Aim 2 we will test the hypothesis that PML contains a DNA-binding domain necessary for recruitment of PML-NBs to viral genomes. Together, these aims will probe the fundamental mechanisms behind the two key events that trigger the formation of PML-NBs in response to HSV infection.