Molecular and cellular mechanisms of HSV-1 assembly and egress

α疱疹病毒，包括重要的人类病原体单纯疱疹病毒1（HSV-1）和人畜共患病原体伪狂犬病病毒（PRV），是极少数已经进化到利用高度专业化的神经元细胞生物学的病毒之一。在疾病的自然过程中，α疱疹病毒感染周围神经系统（PNS）的感觉和自主神经元。在再活化时，子代病毒颗粒在特定的亚细胞位点经历极化的细胞内运输和胞吐作用，以从细胞扩散到细胞，这是本提案的主题。在PNS神经元中，这种细胞内运输可以包括分选成轴突，用于长距离运输到外周组织中，导致疱疹或带状疱疹样病变的复发，或到中枢神经系统（CNS）。人类α疱疹病毒，特别是HSV-1，是病毒性脑炎的主要原因。在目标1中，我们将研究我们的初步数据表明调节非神经元细胞胞内转运的病毒因子，在胞吐作用之前，使用PRV中的现有试剂，并扩展到HSV-1。在目的2中，我们将研究高尔基体后组成性分泌途径机制，其将HSV-1颗粒运输和胞吐引导至非神经元细胞中的特定亚细胞位点，并且我们假设其也介导神经元中的极化运输和胞吐。使用创新的方法，急性扰动特定的细胞因子和直接图像病毒颗粒胞吐，我们将确定分泌途径机制的作用，在细胞内的贩运和出口的病毒颗粒，比较非神经元细胞的主要PNS神经元。在目标3中，我们将专注于微管马达为基础的机制，介导轴突排序，特别是在PNS神经元。使用类似微流体的腔室神经元培养系统和活细胞成像，我们将确定不同驱动蛋白马达和微管相关蛋白在HSV-1颗粒轴突分选中的作用。阐明我们的病毒在神经元和非神经元细胞中使用的基本细胞生物学过程将增加我们对疱疹病毒如何以及为什么传播到神经系统和在神经系统内传播的理解，导致确定可药物靶点和开发更好的病毒神经病理学疗法，并可能提供对细胞生物学的基本见解，特别是神经元的细胞生物学。

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.