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

VZV 潜伏转录本 (VLT) 和 ORF63 在潜伏和重新激活中的作用

• 批准号: 10550430
• 负责人: Paul R. Kinchington
• 金额: $ 72.89万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-17 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550430
• 关键词: Address; Affect; Afferent Neurons; Animal Model; Apoptosis; Autopsy; Binding; Biology; Blindness; Cadaver; Chickenpox; Chromatin; Chronic; Clinical; Code; Detection; Development; Disease; Elderly; Elements; FDA approved; Ganglia; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Herpes zoster disease; Herpesviridae; Herpesvirus Type 3; Histones; Homologous Gene; Human; In Situ; In Vitro; Infection; Intervention; Knowledge; Lytic; Lytic Phase; Lytic Virus; Maintenance; Maps; Methods; Modeling; Modification; Molecular; Mutation; Myocardial Infarction; Neurons; Open Reading Frames; Orthologous Gene; Pain; Phosphorylation; Population; Positioning Attribute; Process; Promoter Regions; Proteins; Quality of life; RNA; RNA Polymerase II; RNA Splicing; Recombinants; Research Personnel; Role; Simplexvirus; Site; Societies; Stroke; Structure of trigeminal ganglion; Testing; Therapeutic; Trans-Activators; Transcript; Vaccines; Viral; Viral Drug Resistance; Viral Genes; Viral Genome; Virus; Virus Diseases; Virus Latency; Work; Zoster Vaccine; chromatin immunoprecipitation; chromatin modification; debilitating pain; design; gene function; genetic regulatory protein; histone methylation; human data; human embryonic stem cell; human pathogen; insight; latency associated transcript; latent infection; mutant; neurotropic; novel; novel therapeutic intervention; promoter; reactivation from latency; viral RNA

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.

水痘-带状疱疹病毒是一种人类阿尔法疱疹病毒(αHV)，在全球90%的人的神经节细胞中建立终身潜伏感染，三分之一重新激活，导致带状疱疹(HZ)、虚弱疼痛和中风。VZV如何维持延迟仍不清楚。尽管有两种FDA批准的HZ疫苗可用，但HZ很可能在未来许多年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抵抗力，重新激活的触发因素仍未明确。我们认为，更好地了解VZV潜伏期和重新激活将为新的抗VZV策略奠定基础，以减轻HZ的负担。目前还没有合适的动物模型来研究VZV的潜伏期和再激活。为了研究这些过程，我们的团队率先开展了自然感染VZV的人三叉神经节(TG)和人类胚胎干细胞(HESC)来源的培养神经元模型的研究。我们发现在人类TG中一致检测到一种新的剪接的VZVRNA，VZV潜伏期相关转录本(VLT)，它的基因组位置与其他嗜神经性αHV相似，并且与开放阅读框架61(ORF61，HSVICP0的直系同源)中的VZV反式激活因子反义。值得注意的是，VLT编码一种功能未知的蛋白质。人TG还含有ORF63RNAs，其蛋白具有调节病毒基因表达和阻断细胞凋亡的作用。我们的假设是，VLT对VZV神经元的感染和潜伏状态是重要的，而ORF63参与了重新激活的启动。我们的三个特定目标旨在研究身体人TG和培养的VZV潜伏期和重新激活的神经元模型中的VZV潜伏期基因。在具体目标1中，我们将对潜伏感染VZV的人TG和感染VZV的培养的hESC神经元进行深入的原位分析，以确定特定的人类神经元亚型是否存在VZV潜伏和重新激活。在具体目标2中，我们将通过制造几个重组VZV VLT突变体并在我们的hESC衍生神经元平台上对它们进行评估，来剖析VLT转录本和/或其编码蛋白对裂解、潜伏和重新激活的VZV感染起作用的机制。最后，特殊目标3将确定VZV潜伏期和重新激活期间VLT和ORF63基因座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3的磷酸/甲基开关和暂停的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，我们现在处于独特的地位，可以在体外表征VZV潜伏期，并在体外系统地分析VLT及其编码蛋白的功能。我们的建议的成功完成将为我们提供对VZV潜伏期和重新激活调节的分子机制的洞察力。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。