Herpes simplex virus egress from cells and spread in neuronal axons

描述(申请人提供)：单纯疱疹病毒(HSV)是一种常见的人类病原体。HSV可在角膜中引发一种称为疱疹间质角膜炎(HSK)的炎性疾病，随着时间的推移，角膜的反复感染会导致角膜基质的进行性疤痕形成。在美国，每年有5万例新发和复发的HSK病例，HSV是导致失明的主要传染病。在上皮或角膜组织中，HSV进入感觉神经元，并通过神经元轴突从外围到达感觉神经节，在那里建立潜伏期。潜伏的单纯疱疹病毒的周期性重新激活会产生病毒颗粒，并将其输送回角膜。HSV在两个方向上的运动包括在微管(MT)上的快速轴突运输。运动复合体沿轴突MT运输细胞蛋白和膜小泡。单纯疱疹病毒和其他a-疱疹病毒已经进化出一种机制，将病毒颗粒拴在这些马达上，使病毒以快速、高度定向的方式从周围神经节移动到神经节，然后再回来。我们的研究将集中在两个HSV膜蛋白，Ge/Gi和US9，促进HSV在神经系统中的定向传播。在动物模型中的研究表明，GE/GI在上皮组织和神经元组织中都起作用，而US9只在神经元中起作用。GE/GI和US9促进HSV的传出运输(从神经节到角膜)，但不促进传入运输(从角膜到神经节)。与此相关，我们使用HSV US9突变体来表征HSK中病毒传播的各个方面。然而，关于HSV gE/gI和US9如何在轴突运输中发挥作用的分子细节知之甚少或一无所知。此外，关于α-疱疹病毒在轴突中的运输形式也存在争议，特别是病毒核衣壳或完全组装的包膜病毒粒子是否向轴突终末运输。我们提出的研究的目的1将检查HSV在培养神经元轴突中的运输，试图更好地理解这一基本的重要过程。HSV Ge/Gi和US9突变体将用于提供病毒在神经细胞体中如何组织或分级的分子细节，病毒的运输形式，以及Ge/Gi和US9如何促进这些过程。目标2将阐述HSV到达轴突末端后发生的步骤。在这个阶段，病毒颗粒必须进入细胞外空间，并穿过神经元和相邻上皮细胞之间形成的连接。这一过程似乎类似于HSV在连接的神经元电路中跨突触传播。我们将研究HSV在体外培养的神经元和上皮细胞之间或连接的神经元之间传播时的这些细胞外事件，并检查病毒膜蛋白如Ge/Gi和US9以及Gb、Gd、Gh/gl的参与。目标3将研究HSV从细胞中排出的更多基本方面，特别是HSV如何穿过核膜。同样，对于核出口和病毒糖蛋白在这一过程中的作用，人们几乎一无所知。抗病毒药物和疫苗的改进设计需要更好地理解单纯疱疹病毒和其他甲型疱疹病毒如何在上皮组织和神经元组织中以高度定向的方式传播。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a common human pathogen. HSV can trigger an inflammatory disease in the cornea, known as herpes stromal keratitis (HSK) in which repeated reinfection of the cornea, over time, leads to progressive scarring in the corneal stroma. There are 50,000 new and recurrent cases of HSK each year in the U.S. and HSV is the leading infectious cause of blindness. In epithelial or corneal tissues, HSV enters sensory neurons and travels in neuronal axons from the periphery to sensory ganglia where latency is established. Periodic reactivation of latent HSV produces virus particles that are transported back to the cornea. HSV movement in both directions involves fast axonal transport on microtubules (MT) over very long distances. Motor complexes ferry cellular proteins and membrane vesicles along axonal MT. HSV and other a-herpesviruses have evolved mechanisms to tether virus particles onto these motors so that the viruses move in a rapid, highly directed fashion from the periphery to ganglia, and back again. Our studies will focus on two HSV membrane proteins, gE/gI and US9, that promote directed spread of HSV in the nervous system. Studies in animal models demonstrated that gE/gI functions in both epithelial and neuronal tissues, while US9 functions exclusively in neurons. gE/gI and US9 facilitate outgoing HSV transport (from ganglia to the cornea) but not incoming transport (from the cornea to ganglia). Related to this, we used an HSV US9 mutant to characterize aspects of viral spread in HSK. However, little or nothing is known about the molecular details of how HSV gE/gI and US9 function in axonal transport. Moreover, there is controversy over the form of a-herpesvirus transport in axons, specifically whether viral nucleocapsids or fully assembled enveloped virions are transported toward axon termini. Aim 1 of our proposed studies will examine HSV transport in axons of cultured neurons, attempting to better understand this fundamentally important process. HSV gE/gI and US9 mutants will be used to provide molecular details of how virus transport is organized or staged in nerve cell bodies, the form of virus that is transported, and how gE/gI and US9 promote these processes. Aim 2 will address steps that occur after HSV reaches the extremities of axons. At this stage, virus particles must move into the extracellular space and across junctions formed between neurons and adjacent epithelial cells. This process appears similar to HSV spread across synapses in connected neuronal circuitry. We will investigate these extracellular events as HSV spreads between cultured neurons and epithelial cells or between connected neurons in vitro, and examine the involvement of viral membrane proteins such as gE/gI and US9, as well as gB, gD, gH/gL. Aim 3 will investigate more basic aspects of HSV egress from cells, specifically how HSV crosses the nuclear envelope. Again, virtually nothing is known about nuclear egress and the role of viral glycoproteins in this process. Improved design of anti-virals and vaccines requires a better understanding of how HSV and other a-herpesviruses spread in a highly directed fashion in both epithelial and neuronal tissues.