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Journal of Virology, November 1999, p. 8989-8998, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Adeno-Associated Virus Type 2 Protein Interactions: Formation of Pre-Encapsidation Complexes

Ralf Dubielzig, Jason A. King, Stefan Weger,dagger Andrea Kern, and Jürgen A. Kleinschmidt*

Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, D-69120 Heidelberg, Germany

Received 2 June 1999/Accepted 2 August 1999

The nonstructural adeno-associated virus type 2 Rep proteins are known to control viral replication and thus provide the single-stranded DNA genomes required for packaging into preformed capsids. In addition, complexes between Rep proteins and capsids have previously been observed in the course of productive infections. Such complexes have been interpreted as genome-linked Rep molecules associated with the capsid upon successful DNA encapsidation. Here we demonstrate via coimmunoprecipitation, cosedimentation, and yeast two-hybrid analyses that the Rep-VP association also occurs in the absence of packageable genomes, suggesting that such complexes could be involved in the preparation of empty capsids for subsequent encapsidation steps. The Rep domain responsible for the observed Rep-VP interactions is situated within amino acids 322 to 482. In the presence of all Rep proteins, Rep52 and, to a lesser extent, Rep78 are most abundantly recovered with capsids, whereas Rep68 and Rep40 vary in association depending on their expression levels. Rep78 and Rep52 are bound to capsids to roughly the same extent as the minor capsid protein VP2. Complexes of Rep78 and Rep52 with capsids differ in their respective detergent stabilities, indicating that they result from different types of interactions. Rep-VP interaction studies suggest that Rep proteins become stably associated with the capsid during the assembly process. Rep-capsid complexes can reach even higher complexity through additional Rep-Rep interactions, which are particularly detergent labile. Coimmunoprecipitation and yeast two-hybrid data demonstrate the interaction of Rep78 with Rep68, of Rep68 with Rep52, and weak interactions of Rep40 with Rep52 and Rep78. We propose that the large complexes arising from these interactions represent intermediates in the DNA packaging pathway.

* Corresponding author. Mailing address: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49-6221-424978. Fax: 49-6221-424962. E-mail: J.Kleinschmidt{at}dkfz-heidelberg.de.

dagger Present address: Institut für Infektionsmedizin, Abteilung Virologie, Freie Universität Berlin, D-12203 Berlin, Germany.

Journal of Virology, November 1999, p. 8989-8998, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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