title: Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells.
abstract: The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University , 313 Ferst Drive, UA Whitaker Bldg, Atlanta, Georgia 30332, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Alonas
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Lifland
authors.forename: Manasa
authors.initials: M
authors.lastname: Gudheti
authors.forename: Daryll
authors.initials: D
authors.lastname: Vanover
authors.forename: Jeenah
authors.initials: J
authors.lastname: Jung
authors.forename: Chiara
authors.initials: C
authors.lastname: Zurla
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kirschman
authors.forename: Vincent F
authors.initials: VF
authors.lastname: Fiore
authors.forename: Alison
authors.initials: A
authors.lastname: Douglas
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Barker
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Wright
authors.forename: James E
authors.initials: JE
authors.lastname: Crowe
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Santangelo
mesh_terms: D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015347:RNA Probes; D060888:Real-Time Polymerase Chain Reaction; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology*; D019065:Virus Assembly
chemical_list: D015347:RNA Probes; D049452:Green Fluorescent Proteins