Alpha herpesviruses, including important human pathogen Herpes Simplex Virus 1 (HSV-1) and zoonotic pathogen Pseudorabies Virus (PRV), are among the very few viruses that have evolved to exploit highly-specialized neuronal cell biology. During the natural course of disease, alpha herpesviruses infect sensory and autonomic neurons of the Peripheral Nervous System (PNS). Upon reactivation, progeny virus particles undergo polarized intracellular trafficking and exocytosis at particular sub-cellular sites to spread from cell to cell, which is the subject of this proposal. In PNS neurons, this intracellular trafficking can include sorting into axons for long-distance transport into peripheral tissues, leading to recurrence of herpetic or zosteriform lesions, or to the Central Nervous System (CNS). The human alpha herpesviruses, and HSV-1 in particular, are leading causes of viral encephalitis. In Aim 1, we will investigate the viral factors that our preliminary data suggest modulate intracellular transport in non-neuronal cells, immediately prior to exocytosis, using existing reagents in PRV, and extending to HSV-1. In Aim 2, we will investigate the post-Golgi constitutive secretory pathway mechanisms that direct HSV-1 particle trafficking and exocytosis to particular sub-cellular sites in non- neuronal cells, and which we hypothesize also mediate polarized trafficking and exocytosis in neurons. Using innovative methods to acutely perturb particular cellular factors and directly image virus particle exocytosis, we will determine the role of secretory pathway mechanisms in intracellular trafficking and egress of virus particles, comparing non-neuronal cells to primary PNS neurons. In Aim 3, we will focus on the microtubule motor-based mechanisms that mediate axonal sorting, specifically in PNS neurons. Using a microfluidics-like chambered neuronal culture system and live-cell imaging, we will determine the roles of different kinesin motors and microtubule-associated proteins in axonal sorting of HSV-1 particles. Elucidating the basic cell biological processes that our viruses use in both neurons and non-neuronal cells will increase our understanding of how and why herpesviruses spread to and within the nervous system, lead to the identification of druggable targets and development of better therapies for viral neuropathology, and may provide fundamental insights into cell biology, particularly of the cell biology of neurons.