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ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2\u00b4-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.<\/jats:p>","DOI":"10.1038\/s41467-024-45842-7","type":"journal-article","created":{"date-parts":[[2024,2,21]],"date-time":"2024-02-21T14:02:42Z","timestamp":1708524162000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Structure of the recombinant RNA polymerase from African Swine Fever Virus"],"prefix":"10.1038","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3251-7376","authenticated-orcid":false,"given":"Simona","family":"Pilotto","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8049-0748","authenticated-orcid":false,"given":"Michal","family":"S\u00fdkora","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7086-8831","authenticated-orcid":false,"given":"Gwenny","family":"Cackett","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2703-7167","authenticated-orcid":false,"given":"Christopher","family":"Dulson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3930-3821","authenticated-orcid":false,"given":"Finn","family":"Werner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,21]]},"reference":[{"key":"45842_CR1","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1038\/nrmicro2507","volume":"9","author":"F Werner","year":"2011","unstructured":"Werner, F. & Grohmann, D. 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