Role of cellular FKBP52 protein in intracellular trafficking of recombinantadeno-associated virus 2 vectors

摘要

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adenoassociatedvirus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 inAAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), andknockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantlyincreased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increasedthe transduction efficiency of conventional AAV vectors by -25-fold in WT MEFs, but only by -4-fold in KO MEFs. The use of selfcomplementaryAAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increasedthe transduction efficiency -23-fold in WT MEFs, but only -4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase inKO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, -59% of AAV genomes werepresent in the nuclear fraction from WT MEFs, but only -28% in KO MEFs, indicating that the pathway by which HU treatment mediates nucleartransport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatmentmediatedincrease in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. IntactAAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. Thesestudies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimaluse of recombinant AAV vectors in human gene therapy.