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Journal of Virology, March 2002, p. 2043-2053, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2043-2053.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors

*** Ziying Yan,1,2 Roman Zak,1,2 G. W. Gant Luxton,1,2 Teresa C. Ritchie,1,2 Ursula Bantel-Schaal,4 and John F. Engelhardt1,2,3*

Department of Anatomy and Cell Biology,1 Department of Internal Medicine,3 Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242,2 Applied Tumor Virology, German Cancer Research Center, Heidelberg, Germany4

Received 3 August 2001/ Accepted 18 November 2001

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.

* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, University of Iowa College of Medicine, 51 Newton Rd., Room 1-111 BSB, Iowa City, IA 52242. Phone: (319) 335-7744. Fax: (319) 335-7198. E-mail: john-engelhardt{at}uiowa.edu.

Journal of Virology, March 2002, p. 2043-2053, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2043-2053.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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