Molecular Genetics of HSV Reactivation

项目总结/摘要 单纯疱疹病毒1型（HSV-1）在外周神经元内建立终身潜伏感染。期间 潜伏期病毒基因组保持为环状附加体，裂解基因沉默。周期性 一些神经元内的基因组重新激活，导致复发性临床疾病。的一大重点 拟议的研究是确定负责调节HSV-1潜伏期的病毒和细胞因子。 在过去的项目期间，三个主要的发现是：1）组蛋白H3 K27 triMe去甲基化酶UTX和 JMJD 3在去除抑制性异染色质组蛋白H3 K27 triMe标记以促进 重新激活此外，我们发现，虽然大多数非终末分化细胞表达UTX和JMJD 3， 组成性地，感觉神经元不这样，但是这些蛋白质由至少一些再激活刺激诱导; 2） 我们鉴定了2个以前未报道的长非编码RNA（TAL和ATL），它们彼此是反义的， 和LAT的5'端。值得注意的是，现有的LAT启动子突变体降低了所有三种转录物的水平 (LAT，TAL和安乐）。此外，我们发现TAL和ATAL转录本在细胞中的表达存在差异。 与表达LAT的神经元仅部分重叠的神经元; 3）我们已经开发了一种方法， 使用AAV载体在体内感觉神经元中敲低病毒和细胞基因。通过击倒LAT 在潜伏期建立后，我们证明了LAT RNA特异性地有助于再激活。在 为了扩展后两个发现，我们提出了以下目标：SA 1：剖析功能角色 新鉴定的LAT区ncRNA TAL和ATAL在调节HSV-1潜伏期和再激活中起作用; SA 2：表征LAT区miRNA在调节HSV-1表型中发挥的功能作用 ，归功于刘备。最后，从我们实验室和其他实验室的工作中可以清楚地看到，HSV潜伏期更长， 动态的，以前赞赏，和潜在的基因表达模式是异质性的。这是 通过发现LAT、TAL和ATAL转录本仅部分重叠表达而突出显示了这一点。 细胞群。因此，在我们的最终目标SA 3中，我们建议使用单细胞RNA-seq分析来鉴定 细胞类型、HSV-1潜伏期和再活化的病毒和宿主基因特征。对于所有这些目标，我们将使用 HSV-1潜伏期和再激活的成熟小鼠和兔模型，但将这些研究扩展到 包括HSV-1潜伏期新的人类神经元模型，并在可能的情况下验证这些发现， 潜伏感染的人类三叉神经节的分析。 这些研究将为细胞和病毒机制提供新的见解， HSV-1潜伏期，并允许将特定功能分配给转录的lncRNA和miRNAs 在LAT地区。这些研究还将提供有关基础的新的关键细节， 潜伏期不同神经元HSV-1基因表达异质性

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.