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Journal of Virology, November 2002, p. 11505-11517, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11505-11517.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Adenovirus-Facilitated Nuclear Translocation of Adeno-Associated Virus Type 2

Wu Xiao,1,2 Kenneth H. Warrington, Jr.,1,3 Patrick Hearing,4 Jeffrey Hughes,1,2 and Nicholas Muzyczka1,3*

Powell Gene Therapy Center,1 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine,3 Department of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, Florida 32610,2 Department of Molecular Genetics and Microbiology, SUNY Stony Brook University, Stony Brook, New York 117944

Received 3 May 2002/ Accepted 31 July 2002

We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex inhibitor thapsigargan. The rapid nuclear translocation of intact AAV capsids in the presence of Ad suggested that one or more Ad capsid proteins might be altering trafficking. Indeed, coinfection with empty Ad capsids also resulted in the appearance of AAV DNA in nuclei within 40 min. Escape from early endosomes did not seem to be affected by Ad coinfection.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, P.O. Box 100266 JHMHSC, Gainesville, FL 32610. Phone: (352) 392-8541. Fax: (352) 392-5914. E-mail: muzyczka{at}ufl.edu.

Journal of Virology, November 2002, p. 11505-11517, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11505-11517.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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