title: Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex.
abstract: Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.
authors.affiliation: Cell Structure Laboratory, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain crisco@cnb.csic.es ifernandez@cnb.csic.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fernández de Castro
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain.
authors.forename: José J
authors.initials: JJ
authors.lastname: Fernández
authors.affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, KY 40546, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Barajas
authors.affiliation: Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, KY 40546, USA.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Nagy
authors.affiliation: Cell Structure Laboratory, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid 28049, Spain crisco@cnb.csic.es ifernandez@cnb.csic.es.
authors.forename: Cristina
authors.initials: C
authors.lastname: Risco
mesh_terms: D000906:Antibodies / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D042541:Intracellular Space / Q000821:virology*; D008668:Metallothionein / Q000378:metabolism; D008954:Models, Biological; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure / Q000821:virology; D017864:Tombusvirus / Q000502:physiology* / Q000648:ultrastructure; D014054:Tomography; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D000906:Antibodies; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D008668:Metallothionein; D012324:RNA-Dependent RNA Polymerase
keywords: 3D electron microscopy; Clonable tag; Metal-tagging transmission electron microscopy; Tomography; Viral replication factory