Herpes Simplex Virus Egress from Cells and Spread into Neuronal Axons

描述(申请人提供)：单纯疱疹病毒1型和2型(HSV)是常见的人类病毒，它在感觉神经节建立终生潜伏期，并周期性地重新激活，重新感染粘膜组织。虽然许多单纯疱疹病毒感染是相对良性的，但角膜的单纯疱疹病毒感染可能会产生严重的后果。单纯疱疹病毒眼部感染会在角膜基质中引发炎症病理，通常是一种免疫屏障组织，产生一种称为疱疹间质角膜炎(HSK)的疾病。HSK经常涉及几个月或几年的反复感染，由重新激活和从神经节向眼睛顺行扩散的循环产生。随着时间的推移，反复感染引起的角膜炎症会造成严重的疤痕和失明。在美国，每年有5万例新发和复发的HSK病例，HSV仍然是导致失明的主要传染病。单纯疱疹病毒与宿主共同进化，使该病毒研究神经系统的时间长达数百万年。在神经元轴突中，HSV搭乘在运动蛋白上，这些蛋白质沿着微管高速公路从神经节中的神经细胞体快速移动到上皮中的轴突尖端。这种快速的轴突运输对于单纯疱疹病毒的存活是必不可少的，以便在完全准备好的宿主面前产生病毒 豁免权，并传播到其他宿主。两种HSV膜蛋白Ge/Gi和US9参与劫持轴突运输机械。GE/GI和US9将作为“分子手柄”，研究HSV在神经元轴突中转运的机制。目的1研究单纯疱疹病毒gE/gI和US9如何促进病毒结构成分的轴突运输。我们发现，缺乏GE/GI和US9的HSV突变体完全阻止了病毒糖蛋白和衣壳向远端轴突的运输。提出了两种机制来解释GE/GI和US9是如何促进轴突运输的。加载机制提示Ge/Gi和US9在神经元胞体中起作用，将HSV结构蛋白分类并加载到微管马达上。接头机制表明，GE/GI和US9在轴突中发挥作用，将病毒蛋白拴在动蛋白马达上。目标2将涉及确定GE/GI和US9中促进装载或适配器功能的分类主题的努力。我们有突变的GE分子，这可能使我们能够区分这两种机制。目的3将通过对轴突中的荧光细胞货物分子进行成像以确定这些分子是否与HSV蛋白共定位，ii)shRNA沉默动蛋白和iii)使用一种涉及分裂动蛋白的新方法来研究哪些动蛋白马达参与了HSV的轴突运输。总之，这些研究将极大地促进我们对HSV如何在神经元轴突中导航的理解，并提供重要的新信息，可用于设计更好的药物或疫苗。

DESCRIPTION (provided by applicant): Herpes simplex viruses types 1 and 2 (HSV) are common human viruses that establish lifelong latency in sensory ganglia and reactivate periodically to reinfect mucosal tissues. While many HSV infections are relatively benign, HSV infections of the cornea can have serious consequences. HSV ocular infections trigger inflammatory pathology in the corneal stroma, normally an immunoprivileged tissue, producing a disease known as herpes stromal keratitis (HSK). HSK frequently involves recurring infections, over months or years, produced by cycles of reactivation and anterograde spread from ganglia to the eye. Over time, inflammation in the cornea caused by repeated reinfections can produce significant scarring and blindness. There are ¿ 50,000 new and recurrent cases of HSK every year in the U.S. and HSV remains the leading infectious cause of blindness. HSV coevolved with its host, allowing millions of years in which the virus has studied the nervous system. In neuronal axons, HSV hitchhikes on motor proteins that move rapidly along microtubule highways from nerve cell bodies in ganglia toward axon tips in epithelium. This fast axonal transport is essential for HSV survival, to allow virus production in the face of fully primed host immunity, and spread to other hosts. Two HSV membrane proteins: gE/gI and US9 are involved in hijacking axon transport machinery. gE/gI and US9 will serve us as "molecular handles" to investigate the poorly understood mechanisms by which HSV is transported in neuronal axons. Aim 1 will investigate how HSV gE/gI and US9 promote axonal transport of viral structural components. We showed that an HSV mutant lacking both gE/gI and US9 was completely blocked in transport of viral glycoproteins and capsids into distal axons. Two mechanisms are proposed to explain how gE/gI and US9 promote axonal transport. The Loading mechanism suggests that gE/gI and US9 function in neuronal cell bodies to sort and load HSV structural proteins onto microtubule motors. The Adaptor mechanism suggests that gE/gI and US9 function in axons to tether viral proteins onto kinesin motors. Aim 2 will involve efforts to identfy sorting motifs in gE/gI and US9 that promote either loading or adaptor functions. We have mutant gE molecules that may allow us to distinguish between these two mechanisms. Aim 3 will investigate which kinesin motors are involved in HSV axonal transport by: i) imaging fluorescent cellular cargo molecules in axons to determine if these are colocalized with HSV proteins, ii) shRNA silencing of kinesins and iii) using a new approach involving split kinesins. Together these studies will substantially advance our understanding of how HSV navigates in neuronal axons and provide important new information that can be used to design better drugs or vaccines.