Molecular and cellular mechanisms of HSV-1 assembly and egress

Molecular and cellular mechanisms of HSV-1 assembly and egress Molecular and cellular mechanisms of HSV-1 assembly and egress Alpha herpesviruses, including important human pathogen Herpes Simplex Virus 1 (HSV-1) and veterinary 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 are 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. The human alpha herpesviruses, and HSV-1 in particular, are leading causes of viral encephalitis, because alpha herpesviruses are also capable of invading the Central Nervous System (CNS). Spread to the CNS can also involve axonal sorting pseudounipolar sensory neurons, and alpha herpesviruses spread to and within the CNS via neuronal synapses. In Aim 1, we will investigate post-Golgi constitutive secretory pathway mechanisms that direct virus 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 2, we will focus on the microtubule motor-based mechanisms that mediate axonal sorting, specifically in PNS neurons. Viral proteins that mediate axonal sorting have been identified in HSV-1 and PRV, and appear to function by recruiting kinesin microtubule motors; although, these viruses by recruit different motors in different ways, so comparing them may be particularly informative. Using a microfluicics-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 vs. PRV. Finally, in Aim 3, we propose an innovative bioengineering goal, to develop methods to directly image virus particle exocytosis at or near neuronal synapses. This will involve culturing primary neurons on patterned substrates in order to induce synapse formation at defined locations and orientations. We will combine this method with our established live-cell virus exocytosis method to be able image and study cell biological factors involved in viral exocytosis specifically and pre- and postsynapses. Elucidating the basic cell biological processes that our viruses use in neurons will increase our understanding of how and why herpesviruses spread in the nervous system, lead to the identification of druggable targets and development of better therapies for viral neuropathology, and may provide fundamental insights into the cell biology of neurons.