title: Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90.
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.
authors.affiliation: Department of Microbiology and Immunology, Walther Oncology Center, Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.forename: Keyun
authors.initials: K
authors.lastname: Qing
authors.forename: Yue
authors.initials: Y
authors.lastname: Si
authors.forename: Linyuan
authors.initials: L
authors.lastname: Chen
authors.forename: Mengqun
authors.initials: M
authors.lastname: Tan
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D016227:Benzoquinones; D015153:Blotting, Western; D004261:DNA Replication; D018076:DNA, Complementary / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D005814:Genes, Viral; D015316:Genetic Therapy; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047029:Lactams, Macrocyclic; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011809:Quinones / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000737:chemistry; D014162:Transfection; D019076:Transgenes; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D016227:Benzoquinones; D018076:DNA, Complementary; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D016376:Oligonucleotides, Antisense; D011809:Quinones; D020032:Tyrphostins; D014443:Tyrosine; D066246:ErbB Receptors; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4; C060641:tyrphostin A23; C001277:geldanamycin