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Journal of Virology, January 2006, p. 810-820, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.810-820.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

Svenja Bleker, Michael Pawlita, and Jürgen A. Kleinschmidt*

German Cancer Research Centre, Infection and Cancer Research Program, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany

Received 4 August 2005/ Accepted 24 October 2005

Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

* Corresponding author. Mailing address: German Cancer Research Centre, Infection and Cancer Research Program, Division of Tumor Virology, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Phone: 49 6221 424978. Fax: 49 6221 42 4962. E-mail: j.kleinschmidt{at}dkfz.de.

Journal of Virology, January 2006, p. 810-820, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.810-820.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • DiPrimio, N., Asokan, A., Govindasamy, L., Agbandje-McKenna, M., Samulski, R. J. (2008). Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly. J. Virol. 82: 5178-5189 [Abstract] [Full Text]  


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