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The disulfide bond of an RGD4C motif inserted within the Hi loop of the adenovirus type 5 fiber protein is critical for retargeting to αv-integrins

Authors

  • Dragomira Majhen,

    1. Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia
    2. University of Paris-Sud, Orsay, France
    3. Vectorologie et Thérapeutiques Anticancéreuses, CNRS UMR 8203, Institut Gustave Roussy, Villejuif, France
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  • Jennifer Richardson,

    1. UMR 1161 Virologie INRA, Anses, ENVA, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France
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  • Bojana Vukelić,

    1. Laboratory of Cellular Biochemistry, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia
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  • Ivana Dodig,

    1. Laboratory for Systems Biomedicine, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
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  • Mario Cindrić,

    1. Laboratory for Systems Biomedicine, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
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  • Karim Benihoud,

    1. University of Paris-Sud, Orsay, France
    2. Vectorologie et Thérapeutiques Anticancéreuses, CNRS UMR 8203, Institut Gustave Roussy, Villejuif, France
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  • Andreja Ambriović-Ristov

    Corresponding author
    • Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia
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A. Ambriović-Ristov, Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia.

E-mail: andrea@irb. hr

Abstract

Background

The αv-integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5.

Methods

Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry.

Results

Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C547–C549 linkage in the C547DC549RGDC553FC555 motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G.

Conclusions

We provide genetic and biochemical data strongly suggesting that cysteines C547 and C549 from the C547DC549RGDC553FC555 motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins. Copyright © 2012 John Wiley & Sons, Ltd.

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