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Journal of Virology, September 2005, p. 11776-11787, Vol. 79, No. 18
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.18.11776-11787.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking{dagger}

Kerstin Lux,1 Nico Goerlitz,1 Stefanie Schlemminger,2 Luca Perabo,1,2 Daniela Goldnau,1,2 Jan Endell,1,2 Kristin Leike,1 David M. Kofler,2 Stefan Finke,3 Michael Hallek,1,2,4 and Hildegard Büning1,2*

Genzentrum, LMU München, Feodor-Lynen-Str. 25, Munich, Germany,1 Klinik I für Innere Medizin, Universität zu Köln, Joseph-Stelzmann-Str. 9, Cologne, Germany,2 Max von Pettenkofer Institut, LMU München, Feodor-Lynen-Str. 25, Munich, Germany,3 GSF-National Center for Research and Environment, Marchioninistr. 25, Munich, Germany4

Received 1 February 2005/ Accepted 14 June 2005

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.

* Corresponding author. Mailing address: University of Cologne, Clinic I for Internal Medicine, LFI, Level 4, Room 052, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. Phone: 49-221-478-4448. Fax: 49-221-478-5455. E-mail: buening{at}lmb.uni-muenchen.de.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, September 2005, p. 11776-11787, Vol. 79, No. 18
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.18.11776-11787.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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