RNA Polymerase II Occupancy and Activity in HSV-Infected Post Mitotic Neurons

在病毒在外周复制后，单纯疱疹病毒（HSV）基因组被递送至感觉神经节的神经元，并在患者的一生中以潜伏状态作为附加体维持。在潜伏期期间，只有1个基因座（LAT）从病毒基因组稳健地转录。这种病毒周期性地重新激活，导致80多个病毒基因表达，并且感染性病毒在上皮表面积累以感染新的个体。在许多个体中，这导致周期性临床疾病，这可能是严重的。本申请试图阐明与人类神经元中HSV潜伏性和生产性感染之间的转录开关相关的机制。具体而言，我们将使用Precision Nuclear Run，然后进行深度测序（PRO-seq）和global run on，然后进行深度测序（GRO-seq），以确定在源自隆德人中脑神经元细胞系（LUHMES）的有丝分裂后神经元中建立静止感染和从静止感染中再活化期间Pol II在病毒基因组上的位置和活性。LUHMES神经元模型首次提供了感染的同步化和足够数量的有丝分裂后神经元，有助于PRO-seq和GRO-seq分析。拟议的研究将测试一个新的假设，Pol II复合物组装和暂停50-80 bp的下游转录起始位点的病毒立即早期基因在静止期。这种启动子近端暂停与超过90%的细胞基因的转录调控一致。在再活化过程中，暂停的复合物被假设伸长到基因体中，允许编码的基因产物的快速产生以启动感染性病毒的产生。虽然数据的高分辨率和定量性质将允许这一假设被明确地测试，它将同时反驳或支持替代假设;例如，病毒附加体是否在静止期间缺乏Pol II，以及重新激活触发Pol II复合物在转录起始位点组装的可能性。我们还将交叉引用数据到已知的抑制性和允许性组蛋白标记的位置，这些标记被认为可以调节LAT表达和其他病毒基因的表达。这一努力将提供关键的洞察力，关于组蛋白介导的沉默的贡献，在控制Pol II定位在静止和再激活。

After virus replication at the periphery, herpes simplex virus (HSV) genomes are delivered to enervating neurons of sensory ganglia, and are maintained as episomes in a latent state for the life of the patient. Only 1 locus (the LAT) is transcribed robustly from the viral genome during latency. The virus reactivates periodically, causing more than 80 viral genes to be expressed, and infectious virus to accumulate at epithelial surfaces to infect new individuals. In many individuals this results in periodic clinical disease, which can be severe. This application seeks to elucidate mechanisms relevant to the transcriptional switches between HSV latent and productive infections in human neurons. Specifically, we will use Precision Nuclear Run followed by deep sequencing (PRO-seq) and global run on followed by deep sequencing (GRO-seq) to establish the position and activity of Pol II on viral genomes during the establishment of, and reactivation from, quiescent infection in post mitotic neurons derived from the Lund Human Mesencephalic neuronal cell line (LUHMES). The LUHMES neuronal model provides, for the first time, both synchronization of infection and sufficient numbers of post- mitotic neurons conducive to PRO-seq and GRO-seq analyses. The proposed study will test a novel hypothesis that Pol II complexes assemble and pause 50-80 bp downstream of transcriptional start sites of viral immediate early genes during quiescence. This promoter proximal pausing is consistent with transcriptional regulation of more than 90% of cellular genes. During reactivation, the paused complexes are hypothesized to elongate into the gene body, allowing rapid production of the encoded gene products to initiate the production of infectious virus. While the data’s high resolution and quantitative nature will allow this hypothesis to be tested unequivocally, it will simultaneously refute or support alternative hypotheses; for example, whether the viral episome is devoid of Pol II during quiescence, and the possibility that reactivation triggers assembly of Pol II complexes at the transcriptional start site. We will also cross reference the data to the already known locations of repressive and permissive histone marks believed to regulate LAT expression and that of other viral genes. This effort will provide key insight regarding the contribution of histone-mediated silencing in the control of Pol II positioning during quiescence and reactivation.