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：
10570901
负责人：
Paul R. Kinchington
金额：
$ 71.33万
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/10570901
关键词：
Ablation 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 Predisposition 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 System 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 nerve stem cell 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.

人类α疱疹病毒（αHV）Varicella-Zoster病毒（VZV）在全球> 90％的人类神经节神经元中建立了终生潜在的潜在感染，在三分之一中重新激活以引起疱疹区域（HZ）（HZ），使疼痛和疼痛和Stroke衰减和Stroke。 VZV如何保持潜伏期尚不清楚。尽管有两种由FDA批准的Hz疫苗可用，但在未来很多年中，HZ可能仍然是一种常见且临床上重要的疾病。目前，VZV潜在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然很差。我们认为，对VZV潜伏期和重新激活的更好了解将为减轻HZ负担的新抗VZV策略建立基础。没有适当的动物模型来研究VZV潜伏期和重新激活。为了研究这些过程，我们的小组开创了对自然VZV感染的人脉神经节（TG）和人类胚胎干细胞（HESC）衍生的培养神经元模型的研究。我们发现了人类TG中新型剪接的VZV RNA的始终检测，即VZV潜伏期相关转录（VLT），该转录的基因组位置与其他神经性αHV中的基因组位置相似，并且与开放式读取框架61（ORF61或HSV ICP0）的VZV反式激活器反义。值得注意的是，VLT编码功能未知的蛋白质。人TG还包含ORF63 RNA，其蛋白质在调节病毒基因表达和阻断凋亡中具有作用。我们上覆的假设是，VLT对于VZV神经元感染和潜在状态很重要，而ORF63参与了重新激活的主动性。我们的三个特定目的旨在解决尸体人类TG和VZV潜伏期和重新激活的培养神经元模型中的VZV潜在基因。在特定的目标1中，我们将对潜在的VZV感染的人类TG和VZV感染的培养hESC神经元进行深入的原位分析，以确定特定的特定人类神经元亚型是否宿主宿主VZV潜伏期和重新激活。在特定目标2中，我们将通过使几个重组VZV VLT突变体在我们的HESC衍生神经元平台中对其进行评估，从而剖析VLT转录本和/或其编码蛋白的机制和/或其编码蛋白质有助于裂解，潜在和重新激活的VZV感染。最后，使用染色质质免疫沉淀，分析CTCF结合，组蛋白蛋白3和RNA Polymase ii rna Polymase ii vzv genom ii n vzv genom n vzv genom n vzv genom on vzv genom ii n vzv genom ii n vzv genom ii n vzv genom penom pen，具体目标3将确定在VZV潜伏期和人类TG和HESC神经元中VLT和ORF63基因座的分子染色质修饰，以分析CTCF的结合，磷酸/甲基甲基蛋白蛋白3和RNA Polna Polymase ii rna Polymeres II On vzv genom On。通过共享我们在VZV生物学方面的完整材料，方法和长期的专业知识，我们现在可以独特地表征VZV潜伏期的离体，并系统地分析VLT及其在体外编码的蛋白质的功能。成功完成我们的建议将洞悉调节VZV潜伏期和重新激活的分子机制。这将为有效针对潜在VZV及其HZ疾病燃烧的新型干预策略的发展提供引导。