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Journal of Virology, December 1999, p. 10010-10019, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Overexpression of Cyclin A Inhibits Augmentation of Recombinant Adeno-Associated Virus Transduction by the Adenovirus E4orf6 Protein

Mirta Grifman,1 Nancie N. Chen,2,dagger Guang-ping Gao,2 Toni Cathomen,1 James M. Wilson,2,3 and Matthew D. Weitzman1,*

Laboratory of Genetics, The Salk Institute for Biological Studies, San Diego, California 92186,1 and Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania Health System,2 and The Wistar Institute,3 Philadelphia, Pennsylvania 19104

Received 24 May 1999/Accepted 27 August 1999

The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.

* Corresponding author. Mailing address: Laboratory of Genetics, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 453-4100, ext. 2037. Fax: (858) 558-7454. E-mail: weitzman{at}salk.edu.

dagger Present address: Department of Microbiology, The University of Hong Kong, Hong Kong, Republic of China.

Journal of Virology, December 1999, p. 10010-10019, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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