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Journal of Virology, March 2005, p. 2831-2838, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2831-2838.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

Philomena Ostapchuk,{dagger} Jihong Yang,{dagger} Ece Auffarth,{ddagger} and Patrick Hearing*

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York

Received 2 September 2004/ Accepted 7 October 2004

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794. Phone: (631) 632-8813. Fax: (631) 632-8891. E-mail: phearing{at}ms.cc.sunysb.edu.

{dagger} P.O. and J.Y. contributed equally to this work.

{ddagger} Present address: Darby and Darby, New York, NY 10022.

Journal of Virology, March 2005, p. 2831-2838, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2831-2838.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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