skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

Abstract

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway bymore » which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

Authors:
;  [1]; ; ;  [1];  [2];  [3];  [4];  [1];  [1]
  1. Div. of Cellular and Molecular Therapy, Dept. of Pediatrics, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610 (United States)
  2. Eli Lilly and Company, Indianapolis, IN 46285 (United States)
  3. Dept. of Microbiology and Immunology, Indiana Univ. School of Medicine, Indianapolis, IN 46202 (United States)
  4. Herman B Wells Center for Pediatric Research and Dept. of Biochemistry and Molecular Biology, Indiana Univ. School of Medicine, Indianapolis, IN 46202 (United States)
Publication Date:
OSTI Identifier:
20850563
Resource Type:
Journal Article
Journal Name:
Virology
Additional Journal Information:
Journal Volume: 353; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2006.04.042; PII: S0042-6822(06)00295-9; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0042-6822
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOSYNTHESIS; DISEASE VECTORS; DNA; EFFICIENCY; EMBRYOS; FIBROBLASTS; GENE THERAPY; GENES; HYDROXYUREA; MICE; PROTEINS; TYROSINE; VIRUSES

Citation Formats

Weihong, Zhao, Jianqing, Wu, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, First Affiliated Hospital of Nanjing Medical Univ., Nanjing, Jiangsu 210029, Li, Zhong, Linyuan, Chen, Weigel-Kelley, Kirsten A, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Keyun, Qing, Larsen, Steven H, Weinian, Shou, Warrington, Kenneth H, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Srivastava, Arun, and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors. United States: N. p., 2006. Web. doi:10.1016/j.virol.2006.04.042.
Weihong, Zhao, Jianqing, Wu, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, First Affiliated Hospital of Nanjing Medical Univ., Nanjing, Jiangsu 210029, Li, Zhong, Linyuan, Chen, Weigel-Kelley, Kirsten A, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Keyun, Qing, Larsen, Steven H, Weinian, Shou, Warrington, Kenneth H, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Srivastava, Arun, & Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors. United States. doi:10.1016/j.virol.2006.04.042.
Weihong, Zhao, Jianqing, Wu, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, First Affiliated Hospital of Nanjing Medical Univ., Nanjing, Jiangsu 210029, Li, Zhong, Linyuan, Chen, Weigel-Kelley, Kirsten A, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Keyun, Qing, Larsen, Steven H, Weinian, Shou, Warrington, Kenneth H, Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610, Srivastava, Arun, and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610. Sat . "Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors". United States. doi:10.1016/j.virol.2006.04.042.
@article{osti_20850563,
title = {Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors},
author = {Weihong, Zhao and Jianqing, Wu and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610 and First Affiliated Hospital of Nanjing Medical Univ., Nanjing, Jiangsu 210029 and Li, Zhong and Linyuan, Chen and Weigel-Kelley, Kirsten A and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610 and Keyun, Qing and Larsen, Steven H and Weinian, Shou and Warrington, Kenneth H and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610 and Srivastava, Arun and Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Univ. of Florida College of Medicine, Gainesville, FL 32610},
abstractNote = {We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.},
doi = {10.1016/j.virol.2006.04.042},
journal = {Virology},
issn = {0042-6822},
number = 2,
volume = 353,
place = {United States},
year = {2006},
month = {9}
}