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

摘要 正如PA-19-237中所强调的，关于非编码RNA及其功能的知识存在空白 对于许多病原体而言，人疱疹病毒水痘带状疱疹病毒（VZV）就是这样一种病原体。VZV是 在人类疱疹病毒中，只有两种病毒的microRNA尚未被报道。我们的搜索 发现了一个新的VZV编码的小非编码RNA（VZVsncRNA）家族，这表明了一个令人兴奋的 VZV可能通过sncRNA调控其感染过程。我们的首要前提是， sncRNA的知识可以形成VZV疾病的新疗法的基础，VZV疾病仍然是一个主要的公众问题。 健康问题。虽然由于水痘疫苗接种，儿童“水痘”在美国不再常见， 大多数成年人在其神经节中仍然潜伏着野生型VZV，具有重新激活的潜力。当它发生时， 带状疱疹的发展，一个痛苦的病态和衰弱的疾病遇到了三分之一，在他们的一生， 经常并发症，最常见的是慢性疼痛。每年仍有约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检测到这些VZVsncRNA。重要的是， 用锁核酸（LNA）抑制剂转染的细胞改变VZV的扩散和生长。在这里我们聚焦 在四种VZVsncRNA上，其与在潜伏感染的人中发现的VZV潜伏转录物（VLT）反义， 神经节和生产性感染，因为LNA抑制剂，他们减少VZV病毒的生产（表明 sncRNA促进感染）。三种LNA抑制剂的组合有效地将VZV生长降低了更多。 超过90%，不仅支持感染的功能作用，而且还建立了一个有前途的新的治疗方法， 战略我们假设这些sncRNA调节上皮细胞的生产性感染，在神经元中， 必须做出的裂解/延迟决策。目的1说明这四种VZVsncRNA如何影响VZV生长 在上皮细胞中使用LNA调节剂，单独和组合，以确定它们如何改变病毒 表达模式为了确定它们是靶向病毒还是宿主，我们将开发改变序列的VZV 编码VZVsncRNA并将它们与表达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，则表明sncRNA可能靶向对VZV重要的宿主途径。我们的主要假设是 该sncRNA靶向VLT RNA以影响关键的促裂解ORF 61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从潜伏期的再激活的假设 在神经元中。我们将利用我们的人类神经元培养系统，该系统拥有一个模型VZV潜伏状态， 我们将开发AAV在潜伏感染的神经元中表达VZVsncRNA， 并确定再活化效率是否有利。在全球范围内，我们的研究将为理解 VZV的新基因调控机制，它们在与VLT相互作用中的可能功能以及VZV的潜在作用。 靶向sncRNA作为治疗带状疱疹的未来治疗方法。

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.