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

 描述（由申请人提供）：非常需要了解人类疱疹病毒水痘带状疱疹病毒（VZV）如何与神经元相互作用。感觉神经元作为水痘带状疱疹病毒长达数十年持续存在的部位，也是水痘带状疱疹病毒重新激活导致带状疱疹的根源，对于水痘带状疱疹病毒的成功发病机制至关重要。三分之一的成年人患有这种痛苦且使人衰弱的疾病，并且经常因慢性疼痛和神经系统问题而变得复杂。此外，眼部带状疱疹是一种潜在的毁灭性传染病，会导致视力严重丧失，并且在疼痛、并发症和生活质量显着破坏方面比其他地方的带状疱疹造成的损失要大得多。即使所有符合条件的人都接种了带状疱疹疫苗（这还远未实现）。仍会产生部分有效性 每年约有 50 万例带状疱疹病例。我们对 VZV 潜伏期和再激活的了解仍然是个谜、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，事实证明，在任何情况下，VZV 实验性重新激活都极其困难。然而，如果可以预防神经元感染、传播、持续或重新激活，那么疾病就可以更容易地得到控制。我们现在拥有一个前所未有的培养人类神经元平台，该平台源自人类胚胎干细胞，不仅支持水痘带状疱疹病毒的生产性感染，而且还支持现在可以重新激活的持久状态。我们最重要的假设是，该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 qreactivation 等方面的问题。我们的首要目标是检验需要特定 VZV 蛋白的假设 用于神经元轴突感染、复制、神经元间传播或顺行轴突返回。我们将评估神经元感染每个阶段的带有基因缺失的荧光报告基因 VZV，以确定所需的蛋白质。此类蛋白质不仅可以成为阻断潜伏期和重新激活策略的目标，而且此类 VZV 突变体还可以成为改进具有明确的向神经性缺陷的疫苗的基础。我们的第二个目标是解决水痘带状疱疹病毒持续存在和重新激活期间的病毒转录程序。然后，我们将确定在持久性和重新激活期间发生的转录，包括寻找可能导致潜伏期的小 RNA。我们还将解决在不同温度下重新激活后转录程序是否不同的问题。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 潜在基因组负载或防止重新激活。这将建立原则或针对潜在的 VZV 基因组，而不依赖于病毒基因产物。在全球范围内，我们的研究将为理解奠定基础 并以以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.