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Journal of Virology, January 2005, p. 28-38, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.28-38.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

John M. Casper, Jennifer M. Timpe, John David Dignam, and James P. Trempe*

Department of Biochemistry and Cancer Biology, Medical College of Ohio, Toledo, Ohio

Received 11 June 2004/ Accepted 3 August 2004

Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5'-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3') was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

* Corresponding author. Mailing address: Department of Biochemistry and Cancer Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Phone: (419) 383-4103. Fax: (419) 383-4131. E-mail: jtrempe{at}mco.edu.

Journal of Virology, January 2005, p. 28-38, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.28-38.2005
Copyright © 2005, 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]  
  • Needham, P. G., Casper, J. M., Kalman-Maltese, V., Verrill, K., Dignam, J. D., Trempe, J. P. (2006). Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro.. J. Virol. 80: 6207-6217 [Abstract] [Full Text]  


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