{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T04:23:58Z","timestamp":1791347038548,"version":"4.3.1"},"posted":{"date-parts":[[2026,1,6]]},"group-title":"Microbiology","reference-count":66,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["R01AI134807"],"award-info":[{"award-number":["R01AI134807"]}]},{"name":"NIH","award":["P30 EY016665"],"award-info":[{"award-number":["P30 EY016665"]}]},{"name":"NIH","award":["2T32EY027721"],"award-info":[{"award-number":["2T32EY027721"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The HSV-1 VP16 protein, delivered to the nuclei of epithelial cells from the tegument layer of infecting virions, drives viral Immediate-Early (IE) transcription to initiate productive (lytic) infection. Latent HSV-1, established in sensory neurons, reactivates to a lytic infection in the absence of a recent\n                  <jats:italic>de novo<\/jats:italic>\n                  infection event that would deliver tegument proteins like VP16. Therefore, the role of the protein VP16 in the exit from HSV-1 latency has been hotly debated for decades. Here we show that VP16 transcription during latency is silenced by CTCF and cohesin proteins bound to a newly identified CTCF insulator site. Upon a reactivation stimulus, CTCF and cohesins are evicted and VP16 is transcribed prior to, and in the absence of, any other viral transcription. We previously identified long-range\n                  <jats:italic>cis<\/jats:italic>\n                  spatial interactions between the LAT and VP16 loci of the latent HSV-1 genome. Here, our data further indicate that the LAT enhancer serves as a neuron-specific enhancer of VP16 transcription during reactivation. Collectively, we show the HSV-1 latent genome is hard-wired for reactivation by a long-range spatial interaction between the viral gene encoding the protein that initiates lytic phase transcription (VP16) and the viral enhancer element that drives its transcription (LAT enhancer).\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>Herpes Simplex Virus 1 (HSV-1) is a significant lifelong human pathogen that infects 70% of adults worldwide. HSV-1 establishes a persistent latent infection in the peripheral nervous system where the virus can periodically reactivate in response to environmental stressors. These reactivation events result in recurrence of corneal infections that can lead to corneal blindness. It is becoming clear that CTCF insulators play a key role in the maintenance of gene silencing in DNA viruses that can establish latency. CTCF insulators are essential regulators of chromatin structure and play vital regulatory roles in transcriptional control of DNA viruses by organizing chromatin architecture during both latent and lytic stages of virus lifecycles. Here we have identified and functionally characterized a novel CTCF insulator, located at the transactivating gene VP16. We provide evidence that this insulator is involved in silencing VP16 during latent infection, while eviction of insulator protein coincides with increased VP16 gene expression during reactivation. To our knowledge, these are the first data that show that CTCF insulators is a key regulatory element in the exit from latency in a VP16-dependent manner and has important implications in understanding the heterogeneity of HSV-1 reactivation in different populations of neurons.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.06.697999","type":"posted-content","created":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T03:15:20Z","timestamp":1767755720000},"source":"Crossref","is-referenced-by-count":1,"title":["In HSV-1, the LAT Enhancer Drives Pre-IE VP16 Transcription to Initiate Reactivation"],"prefix":"10.64898","author":[{"given":"Ziyun A.","family":"Ye","sequence":"first","affiliation":[{"name":"Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America"},{"name":"McPherson Eye Research Institute, University of Wisconsin- Madison, Madison, Wisconsin, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8317-3166","authenticated-orcid":false,"given":"Mason A.","family":"Shipley","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America"},{"name":"McPherson Eye Research Institute, University of Wisconsin- Madison, Madison, Wisconsin, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pankaj","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America"},{"name":"McPherson Eye Research Institute, University of Wisconsin- Madison, Madison, Wisconsin, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8115-2254","authenticated-orcid":false,"given":"Donna M.","family":"Neumann","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America"},{"name":"McPherson Eye Research Institute, University of Wisconsin- Madison, Madison, Wisconsin, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026010710200439000_2026.01.06.697999v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ophtha.2017.05.012"},{"key":"2026010710200439000_2026.01.06.697999v1.2","doi-asserted-by":"publisher","DOI":"10.1097\/00003226-200403000-00001"},{"key":"2026010710200439000_2026.01.06.697999v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2015.11.007"},{"key":"2026010710200439000_2026.01.06.697999v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2015.11.011"},{"key":"2026010710200439000_2026.01.06.697999v1.5","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.04-0614"},{"key":"2026010710200439000_2026.01.06.697999v1.6","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00686-08"},{"key":"2026010710200439000_2026.01.06.697999v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00098-16"},{"key":"2026010710200439000_2026.01.06.697999v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagrm.2009.08.003"},{"key":"2026010710200439000_2026.01.06.697999v1.9","doi-asserted-by":"crossref","unstructured":"Kristie TM . 2015. 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