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Journal of Virology, July 2003, p. 7361-7366, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7361-7366.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Second-Strand Genome Conversion of Adeno-Associated Virus Type 2 (AAV-2) and AAV-5 Is Not Rate Limiting following Apical Infection of Polarized Human Airway Epithelia

Wei Ding,1 Ziying Yan,1 Roman Zak,1 Milene Saavedra,2 David M. Rodman,2 and John F. Engelhardt1*

Department of Anatomy and Cell Biology, Gene Therapy Center for Cystic Fibrosis and Other Genetic Diseases, University of Iowa, Iowa City, Iowa 52242,1 Center for Genetic Lung Disease, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, Colorado 802622

Received 17 December 2002/ Accepted 14 April 2003

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.

* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, Gene Therapy Center for Cystic Fibrosis and Other Genetic Diseases, University of Iowa, Room 1-111, Bowen Science Building, 51 Newton Rd., Iowa City, IA 52242-1109. Phone: (319) 335-7744. Fax: (319) 335-6581. E-mail: john-engelhardt{at}uiowa.edu.

Journal of Virology, July 2003, p. 7361-7366, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7361-7366.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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