Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation.
The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A(1), brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.
Douar
A M
AM
Genethon III-CNRS URA 1923, Evry, France. douar@genethon.fr
Poulard
K
K
Stockholm
D
D
Danos
O
O
D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism / Q000821:virology*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D014771:Virion / Q000378:metabolism
D004279:DNA, Viral; D007976:Leupeptins; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde