dc.contributor.author | Fang, Liang | |
dc.contributor.author | Coutts, Robert H.A. | |
dc.date.accessioned | 2013-11-25T09:29:50Z | |
dc.date.available | 2013-11-25T09:29:50Z | |
dc.date.issued | 2013-11-21 | |
dc.identifier.citation | Fang , L & Coutts , R H A 2013 , ' Investigations on the Tobacco necrosis virus D p60 replicase protein ' , PLoS ONE , vol. 8 , no. 11 , e80912 . https://doi.org/10.1371/journal.pone.0080912 | |
dc.identifier.issn | 1932-6203 | |
dc.identifier.uri | http://hdl.handle.net/2299/12164 | |
dc.description | Copyright: © 2013 Fang, Coutts. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited | |
dc.description.abstract | Tobacco necrosis virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase | en |
dc.format.extent | 10 | |
dc.format.extent | 873771 | |
dc.language.iso | eng | |
dc.relation.ispartof | PLoS ONE | |
dc.title | Investigations on the Tobacco necrosis virus D p60 replicase protein | en |
dc.contributor.institution | Geography, Environment and Agriculture | |
dc.contributor.institution | Crop Protection and Climate Change | |
dc.contributor.institution | Agriculture, Food and Veterinary Sciences | |
dc.contributor.institution | School of Life and Medical Sciences | |
dc.contributor.institution | Health & Human Sciences Research Institute | |
dc.contributor.institution | Department of Human and Environmental Sciences | |
dc.description.status | Peer reviewed | |
rioxxterms.versionofrecord | 10.1371/journal.pone.0080912 | |
rioxxterms.type | Journal Article/Review | |
herts.preservation.rarelyaccessed | true | |