Type I interferons (IFNs) are a powerful family of antiviral cytokines that employ a diverse range of interferon stimulated genes (ISGs) to mediate their effects. As such, many viruses including the betaherpesvirus, human cytomegalovirus (HCMV) employ a variety of IFN and ISG-combatting strategies. This study aimed to evaluate the roles of specific ISGs in regulating HCMV infection and replication. To this end we defined and characterised the IFN-dependent upregulation of nuclear domain 10 (ND10) proteins PML and Sp100 during HCMV infection. The significance of this upregulation was demonstrated through simultaneous and individual ND10 component knockdown. Simultaneous knockdown cells were significantly more permissive to HCMV infection following IFN-β pre-treatment, as were those with individual knockdowns of ISGs PML or Sp100. Individual knockdown of a third ND10 component, hDaxx, whose transcript levels were unaltered during HCMV infection, did not enhance susceptibility to infection following IFN-β pre-treatment. The IFN-dependent transcriptional upregulation of PML and Sp100 was confirmed in our studies using cells engineered to block either the response to or production of IFN through disruption of STAT1 and IRF3 respectively. Investigation of additional ISGs following HCMV infection using these engineered cells indicated that a number of ISGs including ISG15, IFIT1, IFIT2, IFIT3, Mx1, Mx2, and USP18 can be upregulated transcriptionally by both intact and UV-irradiated HCMV independently of interferon but in a manner that is dependent on intact IRF-3 signaling. This IFN signaling independent upregulation of ISGs demonstrates that HCMV and IFN work both separately and together to manipulate ISG expression during infection.