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Journal of Virology, February 2008, p. 1458-1464, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01968-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins{triangledown}

Kevin Nash,* Weijun Chen, and Nicholas Muzyczka

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

Received 7 September 2007/ Accepted 19 November 2007

Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase {delta} as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase {delta}, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase {delta} inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase {delta} play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Florida, 1376 Mowry Rd., Gainesville, FL 32610. Phone: (352) 273 8313. Fax: (352) 273 8284. E-mail: nash{at}ufl.edu

{triangledown} Published ahead of print on 5 December 2007.

Journal of Virology, February 2008, p. 1458-1464, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01968-07
Copyright © 2008, 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]  


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