{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T15:42:53Z","timestamp":1759160573297,"version":"3.37.3"},"reference-count":39,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2015,2,24]],"date-time":"2015-02-24T00:00:00Z","timestamp":1424736000000},"content-version":"vor","delay-in-days":54,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI045000-11A1"],"award-info":[{"award-number":["R01AI045000-11A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["FEBS Open Bio"],"published-print":{"date-parts":[[2015,1]]},"abstract":"<jats:p>Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X\u2010ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside\u20102\u2032\u2010O\u2010methyltransferase (2\u2032\u2010O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2\u2032\u2010O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of<jats:italic>in vitro<\/jats:italic> biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K\u2010D\u2010K\u2010E were sufficient to abolish 2\u2032\u2010O MTase activity<jats:italic>in vitro<\/jats:italic> and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine\u2010N7\u2010methyltransferase activities. Mutations of the surrounding substrate\u2010binding pocket (predicted to recruit cap0) had variable effects on<jats:italic>in vitro<\/jats:italic> VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2\u2032\u2010O MTase function for any member of the<jats:italic>Reoviridae<\/jats:italic> and highlights the significance of K\u2010D\u2010K\u2010E tetrad and surrounding residues for the efficiency of 2\u2032\u2010O MTase activity and in turn, for virus fitness.<\/jats:p>","DOI":"10.1016\/j.fob.2015.02.001","type":"journal-article","created":{"date-parts":[[2015,2,24]],"date-time":"2015-02-24T03:20:10Z","timestamp":1424748010000},"page":"138-146","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Structure\u2010based identification of functional residues in the nucleoside\u20102\u2032\u2010O\u2010methylase domain of Bluetongue virus VP4 capping enzyme"],"prefix":"10.1002","volume":"5","author":[{"given":"Meredith E.","family":"Stewart","sequence":"first","affiliation":[{"name":"Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Polly","family":"Roy","sequence":"additional","affiliation":[{"name":"Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2015,2,24]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000863"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-9-178"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00808-08"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01819-06"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature09489"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2675"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002642"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2010.06.039"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(00)55003-9"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/26694"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(87)90327-8"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20111075"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M800613200"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(92)90600-T"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0509821103"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02806-12"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.03420-13"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1998.1926"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00465-09"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.03571-13"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.15.8059-8067.2004"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00923-14"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.23.13537"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-79-10-2475"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002477"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00814-06"},{"key":"e_1_2_7_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(05)64004-3"},{"key":"e_1_2_7_29_1","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.2008\/002089-0"},{"key":"e_1_2_7_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12013-008-9009-4"},{"key":"e_1_2_7_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2089"},{"key":"e_1_2_7_32_1","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-7-362"},{"key":"e_1_2_7_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(81)90266-X"},{"key":"e_1_2_7_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb1225"},{"key":"e_1_2_7_35_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002698"},{"key":"e_1_2_7_36_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0069825"},{"key":"e_1_2_7_37_1","first-page":"227","article-title":"The effect of temperature on the in vitro transcriptase reaction of bluetongue virus, epizootic haemorrhagic disease virus and African horsesickness virus","volume":"49","author":"Van Dijk A.A.","year":"1982","journal-title":"Onderstepoort J. 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