Project Summary/Abstract Alpha herpesviruses, including the important human pathogen Herpes Simplex Virus 1 (HSV-1), 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 peripheral nervous system (PNS) ganglia, persist as a life-long latent infection, and occasionally reactivate to cause recurrent lesions in peripheral tissues, or can spread to infect the central nervous system. Upon reactivation, replication, and assembly, progeny virus particles can exit from the soma/dendrites of infected neurons, or can sort into axons and undergo long- distance axonal transport. HSV-1, in particular, is a leading cause of viral encephalitis, and may also contribute to the development of neurodegenerative disease. The main objective of this supplement proposal is to determine the microtubule motor-based mechanisms that mediate axonal sorting, specifically in PNS neurons. In Aim 1, we will determine the roles of different kinesin motors and microtubule-associated proteins in intracellular transport of progeny virus particles in non-neuronal cells by live-cell imaging. In Aim 2, we will determine the roles of these motors and microtubule-associated proteins in axonal sorting in primary neurons, using a microfluidics-like chambered neuronal culture system. In this supplement application, the proposed experiments are novel, but are within the scope of the parent R01 award. 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.