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Although DNA polymerase-targeting antivirals are effective, increasing drug resistance underscores the need for alternatives. Helicase\u2013primase inhibitors (HPIs) are promising antivirals, but their mechanisms of action are poorly defined. Furthermore, how the helicase\u2013primase (H\/P) complex and DNA polymerase coordinate genome replication is not well understood for herpesviruses. Here, we report cryo-EM structures of the herpes simplex virus (HSV) H\/P complex bound to HPIs, showing that these lock the helicase-primase complex in an inactive conformation. Single-molecule assays reveal that HPIs cause helicase-primase complexes to pause in unwinding activity on DNA. The structure of an HPI-bound replication fork complex, comprising the H\/P complex (UL5, UL52, and UL8) and polymerase holoenzyme (UL30 and UL42), reveals a previously uncharacterized interface bridging these complexes. These findings provide a structural framework for understanding herpesvirus replisome assembly and advancing inhibitor development.<\/jats:p>","DOI":"10.64898\/2025.12.23.696259","type":"posted-content","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T10:25:10Z","timestamp":1766571910000},"source":"Crossref","is-referenced-by-count":0,"title":["Mechanisms of HSV-1 helicase\u2013primase inhibition and replication fork complex assembly"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6713-3113","authenticated-orcid":false,"given":"Zishuo","family":"Yu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6195-2430","authenticated-orcid":false,"given":"Pradeep","family":"Sathyanarayana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1687-2220","authenticated-orcid":false,"given":"Cong","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2431-0252","authenticated-orcid":false,"given":"Pan","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4519-6276","authenticated-orcid":false,"given":"Sandra K","family":"Weller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8089-8858","authenticated-orcid":false,"given":"Mrinal","family":"Shekhar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2148-5671","authenticated-orcid":false,"given":"Donald M.","family":"Coen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4941-4696","authenticated-orcid":false,"given":"Joseph J.","family":"Loparo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7937-3920","authenticated-orcid":false,"given":"Jonathan","family":"Abraham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025122609550979000_2025.12.23.696259v1.1","unstructured":"Krug, L.T. , and Pellett, P.E . 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