Postentry processing of recombinant adeno-associated virus type 1 and transduction of the ferret lung are altered by a factor in airway secretions.
We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.
Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA 52242-1009, USA.
Ziying
Z
Yan
Xingshen
X
Sun
Idil A
IA
Evans
Scott R
SR
Tyler
Yi
Y
Song
Xiaoming
X
Liu
Hongshu
H
Sui
John F
JF
Engelhardt
D000818:Animals; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D004195:Disease Models, Animal; D005289:Ferrets; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008168:Lung / Q000166:cytology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic; D053586:Virus Internalization / Q000187:drug effects*
D061988:Proteasome Inhibitors; D046988:Proteasome Endopeptidase Complex