title: Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking.
abstract: To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.
authors.affiliation: Genzentrum, LMU München, Munich, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Lux
authors.forename: Nico
authors.initials: N
authors.lastname: Goerlitz
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schlemminger
authors.forename: Luca
authors.initials: L
authors.lastname: Perabo
authors.forename: Daniela
authors.initials: D
authors.lastname: Goldnau
authors.forename: Jan
authors.initials: J
authors.lastname: Endell
authors.forename: Kristin
authors.initials: K
authors.lastname: Leike
authors.forename: David M
authors.initials: DM
authors.lastname: Kofler
authors.forename: Stefan
authors.initials: S
authors.lastname: Finke
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
mesh_terms: D021581:Active Transport, Cell Nucleus; D001483:Base Sequence; D001693:Biological Transport, Active; D002213:Capsid / Q000502:physiology; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000821:virology; D003600:Cytosol / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; C442553:VP2 protein, Dependovirus; D049452:Green Fluorescent Proteins