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Polo\u2010like kinase 1 (PLK1) has been shown to facilitate vaccinia virus (VACV) infection and replication. This study confirms the effects of the PLK1 inhibitors HMN\u2010214 and ON\u201001910 on VACV replication in A549 cells. Both viral titers and DNA loads were significantly reduced in treated cells after infection. Additionally, ON\u201001910 demonstrated broad\u2010spectrum antiviral activity against the lumpy skin disease virus (LSDV) and the infectious bovine rhinotracheitis virus (IBRV) in vitro. PLK1 knockdown in A549 cells also led to a reduction in VACV protein expression, viral titers, and DNA levels. Further analysis showed that VACV infection leads to the accumulation of PLK1 near viral factories. However, despite its strong in vitro effects, ON\u201001910 did not significantly reduce VACV replication in mice. These findings highlight the critical role of PLK1 in VACV replication and its potential as a target for antiviral therapy against orthopoxviruses.<\/jats:p>","DOI":"10.1002\/jmv.70240","type":"journal-article","created":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T13:10:01Z","timestamp":1739625001000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Inhibition of Polo\u2010Like Kinase 1 Dampens the Replication of Vaccinia Virus in Mammalian Cells"],"prefix":"10.1002","volume":"97","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3520-9259","authenticated-orcid":false,"given":"Junda","family":"Zhu","sequence":"first","affiliation":[{"name":"National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine China Agricultural University Beijing 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