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Journal of Virology, June 2007, p. 5777-5787, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02651-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication{triangledown}

Kevin Nash, Weijun Chen, William F. McDonald, Xiaohuai Zhou, and Nicholas Muzyczka*

University of Florida, Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, 1376 Mowry Rd., Gainesville, Florida 32610

Received 30 November 2006/ Accepted 6 March 2007

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase {delta}, but not polymerase {varepsilon} or {alpha}, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase {delta} complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase {delta}, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase {delta}, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Florida, 1376 Mowry Rd., Gainesville, FL 32610. Phone: (352) 273-8114. Fax: (352) 273-8284. E-mail: muzyczka{at}mgm.ufl.edu

{triangledown} Published ahead of print on 14 March 2007.

Journal of Virology, June 2007, p. 5777-5787, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02651-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Guan, W., Wong, S., Zhi, N., Qiu, J. (2009). The Genome of Human Parvovirus B19 Can Replicate in Nonpermissive Cells with the Help of Adenovirus Genes and Produces Infectious Virus. J. Virol. 83: 9541-9553 [Abstract] [Full Text]  
  • Schwartz, R. A., Carson, C. T., Schuberth, C., Weitzman, M. D. (2009). Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase. J. Virol. 83: 6269-6278 [Abstract] [Full Text]  
  • Nash, K., Chen, W., Salganik, M., Muzyczka, N. (2009). Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein. J. Virol. 83: 454-469 [Abstract] [Full Text]  
  • Nash, K., Chen, W., Muzyczka, N. (2008). Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins. J. Virol. 82: 1458-1464 [Abstract] [Full Text]  


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