Journal article
Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors
Journal of virology, Vol.76(5), pp.2043-2053
03/01/2002
DOI: 10.1128/jvi.76.5.2043-2053.2002
PMCID: PMC135943
PMID: 11836382
Abstract
ABSTRACT 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.
Details
- Title: Subtitle
- Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors
- Creators
- Ziying Yan - Department of Anatomy and Cell Biology, Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242Roman Zak - Department of Anatomy and Cell Biology, Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242G. W. Gant Luxton - Department of Anatomy and Cell Biology, Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242Teresa C Ritchie - Department of Anatomy and Cell Biology, Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242Ursula Bantel-Schaal - Applied Tumor Virology, German Cancer Research Center, Heidelberg, GermanyJohn F Engelhardt - Department of Anatomy and Cell Biology, Department of Internal Medicine, Center for Gene Therapy, University of Iowa, Iowa City, Iowa 52242
- Resource Type
- Journal article
- Publication Details
- Journal of virology, Vol.76(5), pp.2043-2053
- DOI
- 10.1128/jvi.76.5.2043-2053.2002
- PMID
- 11836382
- PMCID
- PMC135943
- NLM abbreviation
- J Virol
- ISSN
- 0022-538X
- eISSN
- 1098-5514
- Language
- English
- Date published
- 03/01/2002
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025688802771
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