Adeno-associated virus type 2-mediated gene transfer: Role of cellular FKBP52 protein in transgene expression

Although adeno-associated virus type 2 (AAV) has gained attention as a pote ntially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designat ed the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. K ube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. U SA 94:10879-10884, 1997). We have documented a strong correlation between t he phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. W ang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivasta va, J. Virol. 72:1593-1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kin ase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently , results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, Di. C. Yoder, and A: Srivastava, J. Virol. 72:9835-9841, 1998). Here, we r eport that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppr essant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cD NA. The purified protein could be phosphorylated at both tyrosine and serin e or threonine residues, and only the phosphorylated forms of FKBP52 were s hown to interact with the AAV single-stranded D-sequence probe. Furthermore , in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibi ted AAV second-strand DNA synthesis by greater than 90%. Serine- or threoni ne-phosphorylated FKBP52 caused approximate to 40% inhibition, whereas deph osphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Delib erate overexpression of FKBP52 effectively reduced the extent of tyrosine p hosphorylation of the protein, resulting in a significant increase in AAV-m ediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may ha ve important implications for the optimal use of AAV vectors in human gene therapy.