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

HSV 感染的有丝分裂后神经元中 RNA 聚合酶 II 的占据和活性

• 批准号: 10499255
• 负责人: JOEL D. BAINES
• 金额: $ 7.7万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-06 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10499255
• 关键词: RNA; Polymerase; II; Occupancy; Activity

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.

病毒在周围复制后，单纯疱疹病毒(HSV)的基因组被输送到感觉神经节的衰弱神经元，并在患者的一生中作为潜伏状态的附着体维持。在潜伏期，只有一个基因座(LAT)从病毒基因组中强劲转录。病毒周期性地重新激活，导致80多个病毒基因表达，传染性病毒在上皮表面积聚，感染新的个体。在许多人中，这会导致周期性的临床疾病，这可能是严重的。这项申请旨在阐明与人类神经元中HSV潜伏感染和产生性感染之间的转录开关相关的机制。具体地说，我们将使用精确核运行和深度测序(PRO-SEQ)和全局核运行和深度测序(GRO-SEQ)来确定Pol II在病毒基因组上的位置和活性，在Lund人中脑神经细胞系(LUHMES)的有丝分裂后神经元(LUHMES)的静止感染建立和重新激活期间。LUHMES神经元模型首次提供了感染的同步性和有利于PRO-SEQ和GRO-SEQ分析的足够数量的有丝分裂后神经元。这项拟议的研究将检验一个新的假设，即POL II复合体在静止期间组装并暂停病毒即刻早期基因转录起始点下游的50-80个碱基对。这种启动子近端的停顿与90%以上的细胞基因的转录调控是一致的。在重新激活过程中，暂停的复合体被假设为延伸到基因体中，从而允许编码基因产物的快速生产，以启动传染性病毒的生产。虽然数据的高分辨率和定量性质将使这一假说得到明确的测试，但它将同时驳斥或支持替代假说；例如，病毒Episome在静止期间是否缺乏POL II，以及重新激活触发POL II复合体在转录起始点组装的可能性。我们还将交叉引用已知的抑制性和允许性组蛋白标记的位置，据信这些标记调控LAT和其他病毒基因的表达。这项工作将提供关于组蛋白介导的沉默在静息和重新激活过程中控制POL II定位的作用的关键见解。