title: Analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein.
abstract: Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.
authors.affiliation: INSERM, UMR1043, Toulouse, France.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Charlier
authors.forename: Yuan-Ju
authors.initials: YJ
authors.lastname: Wu
authors.forename: Sophie
authors.initials: S
authors.lastname: Allart
authors.forename: Cécile E
authors.initials: CE
authors.lastname: Malnou
authors.forename: Martin
authors.initials: M
authors.lastname: Schwemmle
authors.forename: Daniel
authors.initials: D
authors.lastname: Gonzalez-Dunia
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001890:Borna Disease / Q000378:metabolism / Q000821:virology*; D001891:Borna disease virus / Q000472:pathogenicity*; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C496537:P protein, Borna disease virus; D010750:Phosphoproteins; D012333:RNA, Messenger; D014760:Viral Fusion Proteins; D015678:Viral Structural Proteins; D049452:Green Fluorescent Proteins; D003545:Cysteine
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