Alpha-herpesviruses, such as human herpes simplex virus (HSV) and varicella-zoster virus (VZV), establish a primary infection at epithelial surfaces, then spread to the neurons of the peripheral nervous system (PNS) where they establish a reactivatable latent infection. Upon reactivation, the viruses spread back to reinfect the epithelial surfaces. On rare occasions, HSV will infect the central nervous system (CNS) to cause encephalitis, often with lethal results. Little is known about the mechanism of neuronal spread and invasion by these viruses. Furthermore, there are plans to harness HSV-1 as a cytotoxic gene delivery vector to target malignant brain tumors. Studies on herpesvirus propagation in the brain might increase the safety and therapeutic effectiveness of this approach. The long-term objective for this research proposal is to understand the molecular mechanisms of viral invasion and spread in the mammalian nervous system-by neurotropic herpesviruses, using pseudorabies virus (PRV) as a model virus. PRV is veterinary virus that is closely related to HSV and VZV and offers numerous technical and experimental advantages for the study of these phenomenas. Previous studies have identified three PRV genes, gE, gI and Us9, required for the efficient directional spread of virus in the rat brain. Viruses defective for any of these genes are impaired in anterograde neuronal spread, suggesting they function to promote viral spread at synapses or in the movement of viral components within neurons. Us9 has been the least studied of these genes and this proposal will focus on the search for neuronal and viral proteins that interact with Us9. Complementary in vivo and in vitro approaches to identify such proteins will be used: (i) yeast two-hybrid selection with cellular and viral gene libraries, (ii) Us9 protein affinity chromatography, and (iii) immunofluorescence localization and immunoprecipitation studies in infected neurons. The discovery of Us9-associated neuronal factors will be of invaluable help in understanding the neurotropic character of these viruses. Similarly, the knowledge of viral proteins interacting with Us9 could allow the identification of additional viral genes involved in neuronal spread, virus assembly, or Us9 intracellular localization.