Molecular Genetics of HSV Reactivation

项目摘要/摘要 单纯疱疹病毒1型(HSV-1)在外周神经元内建立了一种终生潜伏感染。在.期间 潜伏期病毒基因组保持为环状上体，裂解基因沉默。定期 一些神经元内的基因组重新激活，导致反复出现的临床疾病。会议的一个主要焦点是 拟议的研究是为了确定调节HSV-1潜伏期的病毒和细胞因素。 在过去的项目期间，三个主要发现是：1)组蛋白H3K27triMe去甲基酶UTx和 JMJD3在去除抑制性异染色组蛋白H3K27triMe标记中起主要作用 重新激活。此外，我们还发现，虽然大多数非末端分化细胞表达UTX和JMJD3 从结构上讲，感觉神经元不会，但这些蛋白质至少可以被一些重新激活的刺激诱导； 我们鉴定了两个以前未报道的相互反义的长非编码RNA(TAL和ATAL) 和LAT的5‘端。值得注意的是，现有的LAT启动子突变降低了所有三种转录本的水平 (LAT、TAL和ALTAL)。此外，我们还发现TAL和ALT的转录本在 仅与表达LAT的神经元部分重叠的神经元；3)我们已经开发出一种方法 利用AAV载体在体内敲除感觉神经元中的病毒和细胞基因。通过推倒LAT 在建立潜伏期后，我们证明了LAT RNA对重新激活具有特异性。在……里面 为了扩展后两个发现，我们提出了以下目标：SA1：剖析功能角色 新近发现的LAT区ncRNAs、TAL和ALT在调节HSV-1潜伏期和重新激活中发挥作用； SA2：鉴定LAT区miRNAs在调节HSV-1表型中所起的功能作用 归因于LAT。最后，从我们的实验室和其他实验室的工作中可以清楚地看到，HSV延迟更长 以前认识到的动态性，以及潜在的基因表达模式是异质性的。这是 突出的发现是LAT、TAL和ALT的转录本仅部分重叠地表达 大量的细胞。因此，在我们的最终目标SA3中，我们建议使用单细胞rna-seq分析来识别 HSV-1潜伏和重新激活的细胞类型、病毒和宿主基因特征。对于所有这些目标，我们将使用 已经建立了HSV-1潜伏和重新激活的小鼠和兔模型，但将把这些研究扩展到 包括HSV-1潜伏期新人类神经元模型，并在可能的情况下验证这些发现以 人三叉神经节潜伏感染的分析。 拟议的研究将为细胞和病毒调节机制提供新的见解。 HSV-1潜伏期，并允许将特定功能分配给转录的lncRNAs和miRNAs 来自LAT地区。这些研究还将提供关于以下基础的新的关键细节 潜伏期不同神经元HSV-1基因表达的异质性。

Project Summary/Abstract Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection within peripheral neurons. During latency the viral genomes are maintained as circular episomes and the lytic genes are silenced. Periodically the genomes within some of the neurons reactivate resulting in recurrent clinical disease. A major focus of the proposed research is to determine the viral and cellular factors responsible for regulating HSV-1 latency. During the past project period three major findings were: 1) the histone H3K27triMe demethylases UTX and JMJD3 play a major role in removing repressive heterochromatic histone H3K27triMe marks to facilitate reactivation. In addition we found that while most non-terminally differentiated cells express UTX and JMJD3 constitutively, sensory neurons do not, but these proteins are induced by at least some reactivation stimuli; 2) we identified 2 previously un-reported long non-coding RNAs (TAL and ATAL) that are antisense to each other and the 5' end of the LAT. Significantly, existing LAT promoter mutants reduce the levels of all three transcripts (LAT, TAL and ATAL). In addition, we found that the TAL and ATAL transcripts are differentially expressed in neurons with only partial overlap with neurons expressing the LAT; 3) we have developed a means to knockdown viral and cellular genes in sensory neurons in vivo using AAV vectors. By knocking down the LAT after the establishment of latency we demonstrate that the LAT RNA specifically contributes to reactivation. In order to extend the last two of these findings, we propose the following aims: SA1: Dissect the functional roles that the newly identified LAT region ncRNAs TAL and ATAL play in regulating HSV-1 latency and reactivation; SA2: Characterize the functional roles that the LAT region miRNAs play in regulating HSV-1 phenotypes attributed to the LAT. Finally, it is becoming clear from the work of our lab and others that HSV latency is more dynamic that previously appreciated, and that latent gene expression patterns are heterogeneous. This is highlighted by the finding that the LAT, TAL and ATAL transcripts are expressed in only partially overlapping populations of cells. Therefore in our final aim SA3, we propose to use single cell RNA-seq analyses to identify cell type, viral and host gene signatures of HSV-1 latency and reactivation. For all of these aims we will use well-established mouse and rabbit models of HSV-1 latency and reactivation, but will extend these studies to include a novel human neuronal model of HSV-1 latency and validate these findings, where possible, to analyses of latently infected human trigeminal ganglia. The proposed studies will provide novel insights into the cellular and viral mechanisms regulating HSV-1 latency, and allow the assignment of specific functions to the lncRNAs and miRNAs transcribed from the LAT region. These studies will also provide new critical details concerning the basis of heterogeneity of gene HSV-1 gene expression in different neurons during latency.