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J. Biol. Chem., Vol. 279, Issue 13, 12714-12723, March 26, 2004

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Heat-shock Treatment-mediated Increase in Transduction by Recombinant Adeno-associated Virus 2 Vectors Is Independent of the Cellular Heat-shock Protein 90^*

Li Zhong, Keyun Qing, Yue Si, Linyuan Chen, Mengqun Tan¶, and Arun Srivastava||**

From the Department of Microbiology and Immunology, Walther Oncology Center, Walther Cancer Institute, ^||Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, Eli Lilly & Co., Indianapolis, Indiana 46229, and ^¶Central South University, Xiang-Ya School of Medicine, Changsha, Hunan 410078, People's Republic of China

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.

Received for publication, September 24, 2003 , and in revised form, January 6, 2004.

^* This work was supported in part by United States Public Health Service Grants R01 EB-002073 and HL-65570 from the National Institutes of Health (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Indiana University School of Medicine, Medical Science Bldg., Rm. 415-A, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Tel.: 317-274-2194; Fax: 317-274-4090; E-mail: asrivast{at}iupui.edu.

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