Role of VZV Latency Transcript (VLT) and ORF63 in latency and reactivation

水痘带状疱疹病毒（VZV）是一种人类α疱疹病毒（αHV），可在全世界90%的人的神经节神经元中建立终身潜伏感染，在三分之一的人中重新激活，引起带状疱疹（HZ），使人衰弱的疼痛和中风。VZV如何维持延迟尚不清楚。尽管有两种fda批准的赫兹疫苗，但在未来的许多年里，赫兹很可能仍将是一种常见的临床重要疾病。目前，VZV潜伏状态对抗病毒靶向具有抗性，并且重新激活的触发因素仍然不明确。我们认为，更好地了解VZV延迟和再激活将为新的抗VZV策略奠定基础，以减少HZ负担。目前还没有合适的动物模型来研究VZV潜伏期和再激活。为了研究这些过程，我们的团队率先研究了自然感染vzv的人类三叉神经节（TG）和人类胚胎干细胞（hESC）衍生的培养神经元模型。我们发现在人TG中一致检测到一种新的剪接VZV RNA，即VZV潜伏期相关转录物（VLT），其基因组位置与其他嗜神经α hv相似，并且与来自开放阅读框61 （ORF61， HSV ICP0的同源物）的VZV反激活子反义。值得注意的是，VLT编码一种功能未知的蛋白质。人TG也含有ORF63 rna，其蛋白在调节病毒基因表达和阻断细胞凋亡中发挥作用。我们的假设是VLT对VZV神经元感染和潜伏状态很重要，而ORF63参与了再激活的启动。我们的三个特定目标旨在解决VZV潜伏状态基因在尸体人TG和VZV潜伏期和再激活的培养神经元模型中。在Specific Aim 1中，我们将对潜伏VZV感染的人TG和VZV感染的培养hESC神经元进行深入的原位分析，以确定特定的人神经元亚型是否宿主VZV潜伏期和再激活。在Specific Aim 2中，我们将通过制造几种重组VZV VLT突变体并在hesc衍生的神经元平台上评估它们，剖析VLT转录物和/或其编码蛋白对裂解、潜伏和再激活VZV感染的作用机制。最后，Specific Aim 3将确定VZV潜伏期和人TG和hESC神经元再激活期间VLT和ORF63位点发生的分子染色质修饰，使用染色质免疫沉淀分析VZV基因组上CTCF结合、组蛋白蛋白3的磷酸化/甲基化开关和暂停的RNA聚合酶II。通过分享我们在VZV生物学方面的互补材料、方法和长期专业知识，我们现在具有独特的优势，可以表征VZV体外潜伏期，并系统地分析VLT及其编码蛋白的体外功能。本研究的成功完成将为VZV潜伏期和再激活调控的分子机制提供深入的见解。这将为开发新的干预策略提供线索，有效地针对潜伏的VZV，从而减轻HZ疾病的负担。

Varicella-zoster virus (VZV), a human alpha herpesvirus (αHV), establishes lifelong latent infection in ganglionic neurons of >90% humans worldwide, reactivating in one-third to cause herpes zoster (HZ), debilitating pain and stroke. How VZV maintains latency remains unclear. Despite the availability of two FDA-approved HZ vaccines, it is likely that HZ will remain a common and clinically important disease for many years to come. Currently, the VZV latent state is resistant to antiviral targeting, and the triggers of reactivation remain poorly defined. We argue that better knowledge of VZV latency and reactivation will establish the basis for new anti- VZV strategies to reduce the HZ burden. There is no appropriate animal model to study VZV latency and reactivation. To study these processes, our groups pioneered the study of naturally VZV-infected human trigeminal ganglia (TG) and a human embryonic stem cell (hESC)-derived cultured neuron model. We discovered the consistent detection of a novel spliced VZV RNA in human TG, the VZV latency-associated transcript (VLT), which has a genomic position similar to that in other neurotropic αHVs and is antisense to the VZV transactivator from open reading frame 61 (ORF61, ortholog of HSV ICP0). Notably, VLT encodes a protein of unknown function. Human TG also contains ORF63 RNAs, whose protein has roles in regulating viral gene expression and blocking apoptosis. Our overlying hypothesis is that VLT is important for VZV neuronal infection and the latent state, whereas ORF63 is involved in the initiation of reactivation. Our three specific aims are designed to address the VZV latent state genes in both cadaveric human TG and cultured neuron models of VZV latency and reactivation. In Specific Aim 1, we will perform in-depth in situ analyses of latently VZV-infected human TG and VZV-infected cultured hESC neurons to determine if specific human neuron subtypes host VZV latency and reactivation. In Specific Aim 2, we will dissect the mechanisms by which the VLT transcript and/or its encoding protein contribute to lytic, latent and reactivated VZV infections, by making several recombinant VZV VLT mutants and evaluating them in our hESC-derived neuron platform. Finally, Specific Aim 3 will determine the molecular chromatin modifications occurring at the VLT and ORF63 loci during VZV latency and reactivation in both human TG and hESC neurons, using chromatin immunoprecipitations to analyze CTCF binding, phospho/methyl switch of histone protein 3 and paused RNA polymerase II on VZV genomes. By sharing our complementary materials, methods and longstanding expertise in VZV biology, we are now uniquely positioned to characterize VZV latency ex vivo and systematically analyze the function of VLT and its encoded protein in vitro. Successful completion of our proposal will provide insight into molecular mechanisms that regulate VZV latency and reactivation. This will provide leads towards the development of novel intervention strategies that effectively target latent VZV and consequently the burden of HZ disease.