Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors.
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.
Yan
Ziying
Z
Department of Anatomy and Cell Biology, University of Iowa College of Medicine, 51 Newton Road, Iowa City, IA 52242, USA.
Zak
Roman
R
Luxton
G W Gant
GW
Ritchie
Teresa C
TC
Bantel-Schaal
Ursula
U
Engelhardt
John F
JF
D000818:Animals; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D019076:Transgenes; D025801:Ubiquitin / Q000378:metabolism*; D014771:Virion / Q000378:metabolism
D009097:Multienzyme Complexes; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex