Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in sensory neurons and can reactivate from latency under stress conditions. To promote lytic infection, the virus must interact with specific cellular factors to evade the host's antiviral defenses. The HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), activates transcription of viral genes, in part, by mediating the degradation of certain cellular proteins that play a role in host antiviral mechanisms. One component of the cellular defenses that ICP0 disrupts is the suborganelle, nuclear domain 10 (ND10), by inducing the degradation and dissociation of the major organizer of ND10, a promyelocytic leukemia (PML) and ND10 constituent, Sp100. Because previously identified domains in ICP0 explain only partially how it directs the degradation and dissociation of PML and Sp100, we hypothesized that additional regions within ICP0 may contribute to these activities, which in turn facilitate efficient viral replication. To test this hypothesis, we used a series of ICP0 truncation mutants and examined PML protein levels and PML and Sp100 immunofluorescence staining in human embryonic lung cells. Our results demonstrate that two overlapping regions within the central N-terminal portion of ICP0 (residues 212 to 311) promoted the dissociation and degradation of PML and dissociation of Sp100 (residues 212 to 427). In conclusion, we have identified two additional regions in ICP0 involved in altering ND10 antiviral defenses in a cell culture model of HSV-1 infection.