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

基本信息

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
项目类别：
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

项目摘要

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 (VZV) 是一种人类 α 疱疹病毒 (αHV)，可在全球 90% 以上的人类神经节神经元中建立终生潜伏感染，三分之一的人重新激活，导致带状疱疹 (HZ)、使人衰弱的疼痛和中风。尽管有两种 FDA 批准的带状疱疹疫苗可供使用，但带状疱疹很可能在未来许多年内仍将是一种常见且具有重要临床意义的疾病。 VZV 潜伏状态对抗病毒靶向具有抵抗力，并且重新激活的触发因素仍不清楚。我们认为，更好地了解 VZV 潜伏期和重新激活将为减少 HZ 负担的新抗 VZV 策略奠定基础。为了研究 VZV 潜伏期和再激活，我们的团队率先对自然感染 VZV 的人三叉神经节 (TG) 和人胚胎干细胞进行了研究。 (hESC) 衍生的培养神经元模型中，我们发现在人类 TG 中一致检测到一种新型剪接 VZV RNA，即 VZV 潜伏相关转录本 (VLT)，其基因组位置与其他神经营养性 αHV 中的基因组位置相似，并且是开放阅读框 61（ORF61，HSV ICP0 的直系同源物）的反义蛋白，VLT 还编码一种功能未知的蛋白质。 ORF63 RNA 的蛋白质在调节病毒基因表达和阻止细胞凋亡方面发挥作用，我们的首要假设是 VLT 对于 VZV 神经元感染和潜伏状态很重要，而 ORF63 则参与重新激活的启动。解决 VZV 潜伏期和再激活尸体 TG 和培养神经元模型中的 VZV 潜伏状态基因。潜在感染 VZV 的人类 TG 和 VZV 感染的培养 hESC 神经元，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。在特定目标 2 中，我们将剖析 VLT 转录物和/或其编码蛋白促进裂解的机制。、潜伏和重新激活的 VZV 感染，通过制备几种重组 VZV VLT 突变体并在我们的 hESC 衍生神经平台中对其进行评估。最后，Specific Aim 3 将确定人类 TG 和 hESC 神经元中 VZV 潜伏期和重新激活期间 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 CTCF 结合、组蛋白 3 的磷酸/甲基开关和暂停的 RNA通过分享我们在 VZV 生物学方面的补充材料、方法和长期专业知识，我们现在在表征 VZV 潜伏期方面具有独特的优势。体外并系统地分析 VLT 及其编码蛋白的功能，我们的提案的成功完成将深入了解调节 VZV 潜伏和重新激活的分子机制，这将为有效针对潜在 VZV 的新型干预策略的开发提供线索。从而带来带状疱疹疾病的负担。