Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes two important diseases, chickenpox and herpes zoster, separated by a prolonged period of latent infection in which no disease occurs. VZV remains a serious public health concern despite the availability of licensed vaccines for the prevention of chickenpox during childhood and zoster in the elderly. The VZV ORF66 protein kinase is a well-established mediator of VZV tropism and pathogenesis in vivo, but information regarding the molecular mechanisms of ORF66 function is limited. ORF66 is orthologous to the alphaherpesvirus US3 kinase family, which is involved in regulating diverse aspects of the host environment. ORF66 has been implicated in nuclear exclusion of the VZV principle transcriptional regulator and tegument protein, IE62, during late-stage VZV infection; and in downmodulation of class I major histocompatibility complex surface expression, which is critical for the identification and elimination of infected cells by the adaptive immune system. However, the mechanisms underlying these processes are not well-resolved. In this work, I have significantly extended our understanding of known ORF66 functions during VZV infection, and I have established novel functions for both ORF66 and the IE62 protein. Purification of ORF66 allowed the development of an in vitro kinase assay, and I used this to prove that ORF66 directly phosphorylates IE62 adjacent to its nuclear localization signal to mediate nuclear exclusion. Additional investigations of IE62 nucleocytoplasmic trafficking revealed a novel nuclear export activity, which was multi-dimensionally regulated by intrinsic IE62 nuclear retention and phosphorylation by kinase(s) other than ORF66. With regard to the ORF66 role in the regulation of MHC-I surface presentation, I developed assays with recombinant VZV expressing enhanced green fluorescent protein-tagged ORF66 to establish that ORF66 affects the cellular environment such that early MHC-I biogenesis is delayed, resulting in a reduction in total surface MHC-I. MHC-I studies indicated highly novel ORF66 nuclear distribution patterns, and a further examination of these revealed that ORF66 may regulate nucleocapsid morphogenesis or trafficking in VZV-infected nuclei. In sum, this work represents a major contribution to VZV biology and provides insights into a multi-functional mediator of VZV persistence and pathogenesis.