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Journal of Virology, April 2007, p. 3721-3730, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02693-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration{triangledown}

Mary Murphy,{dagger} Janette Gomos-Klein,{dagger},{ddagger} Marko Stankic, and Erik Falck-Pedersen*

Weill Medical College of Cornell University, Hearst Research Foundation, Department of Microbiology and Immunology, Molecular Biology Graduate Program, New York, New York 10021

Received 6 December 2006/ Accepted 24 January 2007

The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

* Corresponding author. Mailing address: Weill Medical College of Cornell University, Department of Microbiology and Immunology Box 62, 1300 York Ave., New York, NY 10021. Phone: (212) 746-6514. Fax: (212) 746-8587. E-mail: efalckp{at}med.cornell.edu

{triangledown} Published ahead of print on 31 January 2007.

{dagger} M.M. and J.G.-K. contributed equally to this work.

{ddagger} Present address: Department of Biological Sciences, City University of New York, Hunter College, New York, NY 10021.

Journal of Virology, April 2007, p. 3721-3730, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02693-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Guilbaud, M., Chadeuf, G., Avolio, F., Francois, A., Moullier, P., Recchia, A., Salvetti, A. (2008). Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration. J. Virol. 82: 2590-2593 [Abstract] [Full Text]  


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