Molecular and cellular mechanisms of HSV-1 assembly and egress

阿尔法疱疹病毒，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)，是极少数进化到利用高度专业化的神经细胞生物学的病毒之一。在自然病程中，阿尔法疱疹病毒感染周围神经系统(PNS)的感觉神经元和自主神经元。在重新激活后，后代病毒颗粒在特定的亚细胞位置经历极化的细胞内运输和胞吐作用，在细胞之间传播，这是本提案的主题。在三叉神经节神经元中，这种细胞内的运输可以包括进入轴突的长距离运输到周围组织，导致疱疹或带状疱疹或带状疱疹的复发，或者到中枢神经系统(CNS)。人类阿尔法疱疹病毒，特别是HSV-1，是病毒性脑炎的主要原因。在目标1中，我们将利用PRV中现有的试剂，研究我们的初步数据表明，在胞吐之前，非神经细胞内的病毒因子调节细胞内转运，并延伸到HSV-1。在目标2中，我们将研究高尔基体后分泌途径机制，该机制将HSV-1颗粒运输和胞吐引导到非神经元细胞中的特定亚细胞位置，我们假设这也介导了神经元中的极化运输和胞吐。使用创新的方法来尖锐地干扰特定的细胞因子和直接成像病毒颗粒的胞吐作用，我们将确定分泌途径机制在病毒颗粒在细胞内运输和输出中的作用，并将非神经元细胞与原代PNS神经元进行比较。在目标3中，我们将重点介绍介导轴突分类的基于微管运动的机制，特别是在三叉神经节神经元。利用微流体室式神经元培养系统和活细胞成像，我们将确定不同的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神经系统内的理解，有助于识别可用药的靶点和开发更好的病毒神经病理学治疗方法，并可能为细胞生物学，特别是神经元的细胞生物学提供基本的见解。

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.