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Journal of Virology, April 2004, p. 3470-3479, Vol. 78, No. 7
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.7.3470-3479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Spacers Increase the Accessibility of Peptide Ligands Linked to the Carboxyl Terminus of Adenovirus Minor Capsid Protein IX

Jort Vellinga,1 Martijn J. W. E. Rabelink,1 Steve J. Cramer,1 Diana J. M. van den Wollenberg,1 Hans Van der Meulen,1 Keith N. Leppard,2 Frits J. Fallaux,1,{dagger} and Rob C. Hoeben1*

Department of Molecular Cell Biology, Leiden University Medical Centre, 2333 AL Leiden, The Netherlands,1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom2

Received 25 June 2003/ Accepted 2 January 2004

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 Å in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

* Corresponding author. Mailing address: Department of Molecular Cell Biology, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Phone: 31-71-5276119. Fax: 31-71-5276284. E-mail: R.C.Hoeben{at}lumc.nl.

{dagger} Present address: Netherlands Institute for Brain Research, 1105 AZ Amsterdam, The Netherlands.

Journal of Virology, April 2004, p. 3470-3479, Vol. 78, No. 7
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.7.3470-3479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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