Abstract |
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 approximately 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.
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Authors | Li Zhong, Keyun Qing, Yue Si, Linyuan Chen, Mengqun Tan, Arun Srivastava |
Journal | The Journal of biological chemistry
(J Biol Chem)
Vol. 279
Issue 13
Pg. 12714-23
(Mar 26 2004)
ISSN: 0021-9258 [Print] United States |
PMID | 14711833
(Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- Benzoquinones
- DNA, Complementary
- HSP90 Heat-Shock Proteins
- Lactams, Macrocyclic
- Oligonucleotides, Antisense
- Quinones
- Tyrphostins
- Tyrosine
- ErbB Receptors
- Tacrolimus Binding Proteins
- tacrolimus binding protein 4
- tyrphostin A23
- geldanamycin
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Topics |
- Benzoquinones
- Blotting, Western
- DNA Replication
- DNA, Complementary
(metabolism)
- Dependovirus
(genetics)
- Down-Regulation
- Electrophoresis, Polyacrylamide Gel
- ErbB Receptors
(metabolism)
- Genes, Viral
- Genetic Therapy
- HSP90 Heat-Shock Proteins
(metabolism)
- HeLa Cells
- Humans
- Lactams, Macrocyclic
- Models, Biological
- Oligonucleotides, Antisense
(chemistry)
- Phosphorylation
- Plasmids
(metabolism)
- Precipitin Tests
- Protein Binding
- Quinones
(pharmacology)
- Tacrolimus Binding Proteins
(chemistry)
- Transfection
- Transgenes
- Tyrosine
(metabolism)
- Tyrphostins
(pharmacology)
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