ABSTRACT Diseases caused by the human herpesvirus Varicella Zoster Virus (VZV) are widespread and debilitating but can be limited by using live attenuated VZV vaccines. The varicella vaccine has been hugely successful in the US, but many countries do not use it widely, some not at all. A high titer version of the same vaccine virus was then developed to immunize adults to boost VZV immunity and reduce the incidence of Herpes Zoster (HZ), the result of VZV reactivation from neuronal latency. HZ is debilitating and complicated, most often by chronic pain that is difficult to treat. HZ remains a public health concern, because most adults harbor wild-type (WT) VZV in their ganglia and are at risk for HZ, and the HZ vaccines have far from optimal coverage in the target populations. The live vaccine virus, vOka, needs improvement. It can cause rashes, go latent and cause rare cases of HZ. It is genetically heterogeneous, with hundreds of single nucleotide polymorphisms (SNPs) occurring at different parent/vaccine allele frequencies. The basis of virus attenuation is not known. 5 SNPs are fully or nearly fully fixed for the vaccine allele and are suspected to direct attenuation. Intriguingly, four lie in the VZV gene encoding IE62, a critical protein that regulates expression of all VZV genes. Excitingly, our data shows that WT VZV, through its IE62, turns on expression of the stem cell epidermal marker KRT15 in keratinocytes and skin, while vaccine virus and its IE62 do not. We then found that KRT15 expression in our epithelial differentiation model is required for VZV replication. Furthermore, KRT15 levels influence the keratinocyte DNA Damage Response (DDR). Taken together, the data support a global hypothesis that IE62 upregulates KRT15 to control pro-viral aspects of the DDR. vOka is attenuated in skin because its IE62 does not trigger the upregulation of KRT15 to regulate DDR pro-viral pathways. To test this hypothesis, Aim 1 will seek to establish that vaccine SNPs in IE62 underlie growth attenuation in models of skin. First, we will use a complementation assay to delineate those vaccine SNPs that prevent IE62 from boosting the replication of vOka vaccine virus in keratinocytes. Second, we will develop WT VZV recombinants that contain ORF62 genes with vaccine SNPs, then quantify their replication in models of skin, including human skin explants. In Aim 2, we will characterize steps of the novel IE62-KRT15-DDR pro-viral pathway that is differentially regulated by KRT15 levels and IE62. This includes studying how the IE62 vaccine genotype influences KRT15 transcription; how KRT15 levels affect the DDR and VZV replication; and what components of the DDR are proviral for VZV in the human epithelial differentiation model. Aim 3 will seek to determine if IE62 specific SNPs underlie the poor reactivation phenotype of vOka from neuronal latency, using cultured human neuron models that have successfully modeled VZV latency and experimental reactivation. T...