title: Live-cell imaging of viral RNA genomes using a Pumilio-based reporter.
abstract: We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.
authors.affiliation: Institute of Molecular Plant Sciences, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK.
authors.forename: Jens
authors.initials: J
authors.lastname: Tilsner
authors.forename: Olga
authors.initials: O
authors.lastname: Linnik
authors.forename: Nynne M
authors.initials: NM
authors.lastname: Christensen
authors.forename: Karen
authors.initials: K
authors.lastname: Bell
authors.forename: Ian M
authors.initials: IM
authors.lastname: Roberts
authors.forename: Christophe
authors.initials: C
authors.lastname: Lacomme
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Oparka
mesh_terms: D005453:Fluorescence; D016679:Genome, Viral; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D017863:Potexvirus / Q000302:isolation & purification; D015202:Protein Engineering; D012367:RNA, Viral / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000648:ultrastructure / Q000821:virology*; D014027:Tobacco Mosaic Virus / Q000302:isolation & purification
chemical_list: C471242:PUM1 protein, human; D012367:RNA, Viral; D016601:RNA-Binding Proteins