Molecular studies of VZV infection, latency and reactivation in human neurons in-vitro

 描述（由申请人提供）：非常需要了解人疱疹病毒水痘带状疱疹病毒（VZV）如何与神经元相互作用。感觉神经元是VZV成功发病的关键，因为它是持续数十年的持续状态的部位，也是VZV重新激活导致带状疱疹的来源。三分之一的成年人都会遇到这种痛苦和衰弱的疾病，并且经常伴有慢性疼痛和神经问题。此外，眼部带状疱疹是一种潜在的破坏性传染病，其导致显著的视力丧失，并且在疼痛、并发症和生活质量的显著破坏方面比其他地方的带状疱疹造成更大的死亡。即使所有符合条件的人都接种了带状疱疹疫苗（这还远远没有实现）。仍然会产生部分效力， 每年约有50万带状疱疹病例我们所知道的VZV潜伏期和再激活仍然是谜，有争议和不清楚，因为大多数动物模型或其神经组织不支持VZV复制或再激活。事实上，VZV的实验性再激活在任何情况下都被证明是极其困难的。然而，如果可以预防神经元感染、扩散、持续或重新激活，疾病就可以更容易地控制。我们现在有了一个前所未有的培养人类神经元平台，来自人类胚胎干细胞，不仅支持VZV生产性感染，而且支持现在可以重新激活的持久状态。我们的假设是，这个系统将使我们能够以一种以前不可能的方式解决神经元感染、潜伏期和再激活的问题。我们的第一个目标将测试特定VZV蛋白是必需的假设， 用于神经元轴突感染、复制、神经元间扩散或顺行轴突返回。我们将评估荧光报告VZV基因缺失的神经元感染的每个阶段，以确定这些蛋白质所需的。这些蛋白质不仅可以在阻断潜伏期和再激活的策略中被靶向，而且这些VZV突变体可以成为具有确定的嗜神经性缺陷的改进疫苗的基础。我们的第二个目标将解决VZV持久性和再激活过程中的病毒转录程序。然后，我们将确定在持久性和再激活过程中发生了什么转录，包括寻找可能导致延迟的小RNA。我们还将解决，如果转录程序不同后，在室温下重新激活。我们的第三个目标将测试以下假设：持久性VZV基因组可以被大序列识别核酸酶靶向，以减少VZV潜在基因组负荷或防止再激活。这将确立不依赖病毒基因产物而靶向潜伏VZV基因组的原则。在全球范围内，我们的研究将为理解 并以先前不可能的方式瞄准VZV潜伏状态。

 DESCRIPTION (provided by applicant): There exists a great need to understand how the human herpesvirus Varicella zoster Virus (VZV) interacts with neurons. The sensory neuron is critical to successful VZV pathogenesis as the site of a decades-long state of persistence, and the source for VZV reactivation to cause Herpes zoster. This painful and debilitating disease is encountered by a third of adults, and is frequently complicated by chronic pain and neurological problems. Furthermore, ophthalmic zoster is a potentially devastating infectious disease that causes significant vision loss and a much greater toll than zoster elsewhere in terms of pain, complications and marked disruption of the quality of life. Even if all eligible persons received the zoster vaccine (which is far from being achieved). the partial effectiveness would still result in some half of a million zoster cases annually. What we know of VZV latency and reactivation has remained enigmatic, controversial and unclear, because most animal models or their neurological tissues do not support VZV replication or reactivation. Indeed, VZV experimental reactivation has proved exceedingly difficult under any circumstances. Yet, if neuronal infection, spread, persistence or reactivation can be prevented, disease could be more easily controlled. We now have an unprecedented cultured human neuron platform, derived from human embryonic stem cells, that supports not only VZV productive infection, but also persistent states that can now be reactivated. Our overlying hypothesis is that this system will enable us to address aspects of neuron infection, latency and qreactivation in a manner that has not been previously possible. Our first aim will test the hypothesis that specific VZV proteins are required for neuron axonal infection, replication, inter-neuronal spread or anterograde axonal return. We will evaluate fluorescent reporter VZV with gene deletions for each stage of neuronal infection to identify those proteins required. Such proteins could be not only be targeted in strategies to block latency and reactivation, but such VZV mutants could be the basis for improved vaccines with defined deficiencies in neurotropism. Our second aim will address the viral transcription program during VZV persistence and reactivation. We will then identify what transcription occurs during persistence and reactivation, including a search for small RNAs that may contribute to latency. We will also address if transcriptional programs differ following reactivation at differen temperatures. Our third aim will test the hypothesis that the persistent VZV genome can be targeted by large sequence recognition nucleases, in order to reduce VZV latent genomic loads or prevent reactivation. This would establish principles or targeting the latent VZV genome without reliance on viral gene products. Globally, our studies will set the stage for understanding and targeting the VZV latent state in a manner that has not been previously possible.