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 attachmen t to a variety of cell surface receptors (heparan sulfate, integrins, and f ibroblast growth factor receptor 1) followed by clathrin-dependent or indep endent internalization. Here we have studied the subsequent intracellular t rafficking of AAV particles from the endosomal compartment to the nucleus, Human cell lines were transduced with a recombinant AAV (rAAV) carrying a r eporter gene (luciferase or green fluorescent protein) in the presence of a gents that affect trafficking. The effects of bafilomycin A(1), brefeldin A , and MG-132 were measured. These drugs act at the level of endosome acidif ication, early-to-late endosome transition, and proteasome activity, respec tively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different fr om 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 facilita tes the conversion of the rAAV single-stranded DNA genome into its replicat ive form, MG-132, a drug currently in clinical use, could be of practical u se for potentializing rAAV-mediated delivery of therapeutic genes.