Varicella-zoster virus (VZV) is an evolutionarily ancient alphaherpesvirus with a 125 kbp genome. VZV causes two diseases -- chickenpox and shingles; the latter disease is a remarkable illustration of VZV neurotropism and reactivation from a prolonged latency. The goal of the current proposal is an increased understanding at a molecular level of the structure/function relationships of the two unique short glycoproteins called gE and gI which form a Fc receptor complex. The herpesviral gE/gI complex is known to be an important determinant of viral egress and cell-to-cell spread, but the mechanisms are not well understood. The Research Plan contains three Specific Aims. Aim 1 includes a characterization of the phosphorylation and sorting motifs in the C-tail of gE. The glycoprotein receptor is modified by both serine and tyrosine protein kinases; tyrosine phosphorylation occurs only on a dimeric form of gE and has not been previously recognized. Phosphorylation will be measured by both in vivo and in vitro protein kinase assays. The fact that both serine and tyrosine phosphorylation motifs are common features of several mammalian cell surface receptors supports the hypothesis that VZVgE/gI form a pluripotential receptor complex. Aim 2 seeks through a mutagenesis approach to further identify the serine protein kinase which phosphorylates the unusual serine-proline-proline sequence in the C-tail of gI, and also identify internalization motifs in proximity to the phosphorylation site. In Aim 3, the interaction of the two components of the gE/gI complex will be analyzed before and after mutagenesis in order to determine the specific roles of each signaling motif on the function of the entire complex. Endocytosis and recycling of the VZV gE/gI complex will be investigated in detail. As a complementary strategy to transient transfection assays, recombinant VZV gE-pseudorabies viruses will be produced and evaluated in an animal model of neurotropism, and VZV mutants with a gI null phenotype will be investigated by several imaging techniques, including laser scanning confocal microscopy and electron microscopy with immunolabeling. In summary, the phosphorylation modifications of VZV gE/gI support an evolutionary link with other nonviral receptors and, at the same time, suggest a role for phosphorylation-dephosphorylation events in trafficking and endocytic pathways involved in viral egress and cell-to-cell spread.