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 postmitotic 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.