Intersection of HSV latency and reactivation with the neuronal apoptotic pathway

描述(申请人提供)：单纯疱疹病毒(HSV)以潜伏感染神经元的形式在宿主的一生中持续存在。重要的是，VUS的周期性重新激活会导致显著的发病率和死亡率，尤其是在免疫功能低下的宿主中。然而，允许病毒持续和重新激活的神经元和分子事件的独特特征尚不清楚。到目前为止，理解病毒与神经元相互作用的障碍是：1)潜伏期体外模型的有限使用；2)初级神经元基因操纵技术的挑战。已发现交感神经元感染可在体内重现HSV潜伏期。因此，在这个项目中，我将使用交感神经元和最先进的技术来检测病毒基因功能，以及在分子和细胞水平上病毒在神经元中重新激活的机制。潜伏期相关转录本(LAT)编码一系列非编码RNA，是唯一在潜伏期高水平表达的病毒基因产物。在目标1中，我将测试LAT表达抑制细胞凋亡和促进受感染神经元存活的假设，从而允许病毒长期存在和增强再激活。随后将通过显微注射将表达质粒导入神经元。还将确定LAT保护神经元免受不同凋亡触发因素的能力，以及LAT发挥保护作用的机制。在目标2中，我将重点研究触发HSV重新激活的神经元内的信号事件。当交感神经元失去神经生长因子(NGF)时，病毒重新激活。由于NGF剥夺激活了神经元的凋亡，我将确定NGF剥夺后的凋亡途径中的关键事件，它激活HSV裂解基因的表达，从而允许病毒重新激活。了解HSV潜伏期是如何在细胞水平上维持的，以及神经元内触发HSV重新激活的关键事件的知识，对于识别防止HSV从神经元重新激活的新疗法的潜在靶点至关重要。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) persists for the lifetime of the host in the form of a latent infection of neurons. Importantly, periodic reactivation of the vius results in significant morbidity and mortality, particularly in the immunocompromised host. However, the unique characteristics of neurons and molecular events that allow viral persistence and its reactivation are not understood. So far, obstacles to understanding the interaction of the virus with neurons have been i) the limited use of an in vitro model of latency and ii) challenges in gene manipulation techniques in primary neurons. Infection of sympathetic neurons has been found to recapitulate HSV latency in vivo. Therefore, in this project I will use sympathetic neurons and state-of-art techniques to examine viral gene function, and the mechanism of viral reactivation at the molecular and cellular level in neurons. The latency-associated transcript (LAT) encodes a family of non-coding RNAs and is the only viral gene product expressed to high levels during latency. In Aim 1, I will test the hypothesis that LAT expression inhibits apoptosis and promotes survival of infected neurons, thus allowing for long-term viral persistence and enhanced reactivation. LAT expressing plasmids will be introduced into neurons by microinjection. The ability of the LAT to protect neurons against different triggers of apoptosis and the mechanism by which the LAT exerts protection will also be determined. In Aim 2, I will focus on examining the signaling events within neurons that trigger HSV reactivation. Viral reactivation is triggered when sympathetic neurons are deprived of nerve growth factor (NGF). Since NGF deprivation activates apoptosis in neurons, I will identify the key event in the apoptotic pathway after NGF deprivation that activates the expression of HSV lytic genes to allow viral reactivation. An understanding of how HSV latency is maintained at the cellular level and knowledge of key events within neurons that trigger its reactivation are critica to identify potential targets for novel therapeutics that prevent HSV reactivation from neurons.