Small non-coding RNAs of VZV: role in lytic and latent infection

VZV 小非编码 RNA：在裂解和潜伏感染中的作用

• 批准号: 10310489
• 负责人: Ronald Goldstein
• 金额: $ 20.86万
• 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: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10310489
• 关键词: Acids; Address; Adult; Affect; Agonist; Antisense RNA; Cadaver; Cell Line; Cells; Cellular Immunity; Chickenpox; Childhood; Chronic; Climacteric; Development; Disease; Elderly; Elements; Epithelial; Epithelial Cells; Family; Frequencies; Future; Ganglia; Gene Expression; Genes; Genetic Transcription; Goals; Growth; Herpes zoster disease; Herpesviridae; Herpesvirus Type 3; Human; Impairment; Infection; Knowledge; Lytic; Lytic Phase; Messenger RNA; MicroRNAs; Modeling; Mutate; Mutation; Nervous system structure; Neurons; Nucleotides; Oligonucleotides; Pain; Parents; Pathway interactions; Patients; Pattern; Persons; Process; Production; Proteins; Public Health; RNA; Regulator Genes; Reporting; Risk; Role; Small RNA; System; Testing; Therapeutic; Transcript; Transfection; Untranslated RNA; Vaccination; Viral; Viral Genome; Virus; Virus Diseases; Virus Latency; Virus Replication; Work; Zoster Vaccine; adeno-associated viral vector; chronic pain; embryonic stem cell; genetic regulatory protein; inhibitor; interest; latent infection; locked nucleic acid; mutant; neuronal growth; novel; novel therapeutic intervention; novel therapeutics; nucleic acid inhibitor; pathogen; prevent; reactivation from latency; transcriptome

Abstract As highlighted in PA-19-237, there is a gap in knowledge about non-coding RNAs and their functions for many pathogens, and the human herpesvirus Varicella Zoster Virus (VZV) is one such pathogen. VZV is one of only two of nine human herpesviruses in which microRNAs have not been reported. Our searches identified a family of novel VZV-encoded small non-coding RNAs (VZVsncRNA), suggesting an exciting possibility that VZV regulates its infectious process using sncRNA. Our overarching premise is that better knowledge of sncRNA could form the basis for novel therapies for VZV disease, which is still a major public health concern. While childhood “chickenpox” is no longer common in the US due to varicella vaccination, most adults still harbor wild-type VZV in their ganglia in a latent state, with potential to reactivate. When it does, Herpes Zoster develops, a painful morbid and debilitating disease encountered by a third in their lifetime that is frequently complicated, most often by chronic pain. There is still some half of a million HZ cases annually. We detected these VZVsncRNA in both infected epithelial cells and lytic infected neurons. Importantly, cells transfected with locked nucleic acid (LNA) inhibitors to them alter VZV spread and growth. Here we focus on four VZVsncRNA which are antisense to the VZV Latency Transcript (VLT) found in latently infected human ganglia and productive infections, as LNA inhibitors to them reduce VZV virus production (indicating the sncRNA promote infection). A combination of three LNA inhibitors effectively reduced VZV growth by more than 90%, not only supporting a functional role in infection but also establishing a promising novel therapeutic strategy. We hypothesize that these sncRNA regulate epithelial productive infections and in neurons, the lytic/latency decisions that must be made. Aim 1 addresses how these four VZVsncRNA influence VZV growth in epithelial cells using LNA modulators, in single and in combination, to determine how they change viral expression patterns. To determine if they target virus or host, we will develop VZV altered in sequence encoding the VZVsncRNA and using them in conjunction with epithelial cell lines expressing the VZVsncRNA. Our hypothesis predicts that specific virus mutation will prevent expression of sncRNA from influencing VZV infection; if not, it will indicate sncRNA may target host pathways important for VZV. Our primary hypothesis is that sncRNA target VLT RNA to influence expression of the critical pro-lytic ORF61 protein. Our second aim will test the hypotheses that the four VZVsncRNA promote lytic infection or enhance reactivation from latency in neurons. We will exploit our human neuron culture system that hosts a model VZV latent state and experimental reactivation from it. We will develop AAV to express VZVsncRNA in latently infected neurons and determine if reactivation efficiency is favored. Globally, our studies will set the stage for understanding a novel gene regulatory mechanism for VZV, their possible functions in interacting with VLT and the potential for targeting sncRNA as a future therapeutic approach for treating Herpes zoster.

摘要 正如PA-19-237中强调的那样，关于非编码RNA及其功能的知识存在缺口 对于许多病原体来说，人类疱疹病毒水痘带状疱疹病毒(VZV)就是这样的病原体之一。VZV是 九种尚未发现微小RNA的人类疱疹病毒中仅有的两种之一。我们的搜索 发现了一族新的VZV编码的小非编码RNA(VZVsncRNA)，暗示了一种令人兴奋的 VZV可能通过SNcRNA调控其感染过程。我们最重要的前提是 对SncRNA的了解可以为VZV病的新疗法奠定基础，VZV病仍然是一种主要的公众疾病 健康问题。虽然由于接种了水痘疫苗，儿童时期的水痘在美国不再常见， 大多数成年人仍在神经节中潜伏着野生型VZV，有可能重新激活。当它发生的时候， 带状疱疹是一种令人痛苦的病态和衰弱的疾病，三分之一的人一生中会遇到这种疾病 经常是复杂的，最常见的是慢性疼痛。每年仍有大约50万例HZ病例。 我们在感染的上皮细胞和裂解感染的神经元中都检测到了这些VZVsncRNA。重要的是 转染锁定核酸(LNA)抑制剂的细胞可以改变VZV的传播和生长。在这里，我们专注于 人潜伏感染VZV潜伏转录本(VLT)反义VZVsncRNA的研究 神经节和生产性感染，因为LNA抑制剂可以减少VZV病毒的产生(表明 SncRNA促进感染)。三种LNA抑制剂的组合有效地抑制了VZV的生长 超过90%，不仅支持在感染中发挥作用，而且还建立了一种有前景的新疗法 策略。我们假设这些SNcRNA调节上皮性感染，在神经元中， 必须做出的分解/延迟决策。目标1阐述这四种VZVsncRNA如何影响VZV生长 在上皮细胞中使用LNA调节剂，单独和联合使用，以确定它们如何改变病毒 表情模式。为了确定它们是针对病毒还是针对宿主，我们将按顺序开发改变的VZV 编码VZVsncRNA，并将其与表达VZVsncRNA的上皮细胞系结合使用。 我们的假设预测，特定的病毒突变将阻止SncRNA的表达影响VZV 感染；如果不是，这将表明SncRNA可能针对对VZV重要的宿主途径。我们的主要假设是 SncRNA以VLT RNA为靶标，影响关键的ORF61蛋白的表达。我们的第二个目标 将检验四种VZVSncRNA促进裂解性感染或增强潜伏期重新激活的假设 在神经元中。我们将开发我们的人类神经元培养系统，该系统拥有VZV模型潜伏期和 从它身上进行实验性的再激活。我们将开发AAV在潜伏感染的神经元中表达VZVsncRNA 并确定是否有利于重新激活效率。在全球范围内，我们的研究将为理解 VZV新的基因调控机制及其与VLT相互作用的可能功能 靶向SncRNA作为未来治疗带状疱疹的方法。