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Journal of Virology, June 2003, p. 6394-6404, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6394-6404.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhancement of Recombinant Adeno-Associated Virus Type 2-Mediated Transgene Expression in a Lung Epithelial Cell Line by Inhibition of the Epidermal Growth Factor Receptor

Andrew D. Smith, Roy F. Collaco, and James P. Trempe*

Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804

Received 16 December 2002/ Accepted 10 March 2003

Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as gene delivery systems because they show long-term expression in vivo and transduce numerous cell types. Limitations to successful gene transduction from rAAVs have prompted investigations of a variety of treatments to enhance transgene expression from rAAV vectors. Tyrphostin-1, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, dramatically enhances rAAV transgene expression. Elegant studies have demonstrated that a single-strand D-sequence-binding protein (ssDBP) is phosphorylated by EGFR and binds to the D sequence element in the AAV terminal repeat (TR). Binding of the Tyr-phosphorylated ssDBP prevents conversion of single-stranded vector DNA to a double-strand conformation. We observed dramatic increases in transgene expression in lung epithelial cells (IB3) with tyrphostin treatment. Gel shift analysis of ssDBP revealed that its DNA binding characteristics were unchanged after tyrphostin treatment or adenovirus infection. Tyrphostin stimulated rAAV transgene expression to a greater extent than adenovirus coinfection. Southern hybridizations revealed that the vector DNA remained in the single-strand conformation in tyrphostin-treated cells but double-stranded replicative form monomer DNA was most abundant in adenovirus-infected cells. Northern analyses revealed that tyrphostin treatment enhanced mRNA accumulation more than in adenovirus-infected cultures even though replicative form DNA was undetectable. Analysis of the JNK, ERK, and p38K mitogen-activated protein kinase pathways revealed that tyrphostin treatment stimulated the activity of JNK and p38K. Our data suggest that tyrphostin-induced alteration of stress response pathways results in dramatic enhancement of transcription on linear vector DNA templates in the IB3 cell line. These results expand the downstream targets of the EGFR in regulating rAAV transduction.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Phone: (419) 383-4103. Fax: (419) 383-6228. E-mail: JTREMPE{at}MCO.EDU.

Journal of Virology, June 2003, p. 6394-6404, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6394-6404.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Timpe, J. M., Verrill, K. C., Trempe, J. P. (2006). Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection.. J. Virol. 80: 7807-7815 [Abstract] [Full Text]  
  • Sugano, E., Tomita, H., Ishiguro, S.-i., Abe, T., Tamai, M. (2005). Establishment of Effective Methods for Transducing Genes into Iris Pigment Epithelial Cells by Using Adeno-associated Virus Type 2. IOVS 46: 3341-3348 [Abstract] [Full Text]  


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