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Cysteines surrounding an RGD motif inserted in the HI loop of human adenovirus type 5 fiber protein influence its retargeting potential

Absence of coxsackie-adenovirus receptor (CAR) on the cell surface, or its suboptimal localization, markedly impedes human adenovirus type 5 (Ad5) infection. One of the possible approaches to solve this obstacle is adenovirus retargeting by incorporation of retargeting motifs in the HI-loop of the Ad5 fiber protein. Several retargeting motifs have been evaluated ; however, RGD4C, an α v-integrin binding peptide that enhanced infection of a wide range of tumor cells in vitro and in vivo, has been used most extensively. The RGD4C peptide contains two pairs of cysteine residues (CDCRGDCFC), introduced in an attempt to stabilize the sequence via formation of disulfide bonds between the cysteines. It should be emphasized that an RGD motif without cysteines (MNDHAIRRGDTFATTRAE) is naturally present within the penton base of wild type Ad5. Both of these RGD containing motifs, potentially cyclic or linear ; have been inserted into the HI loop of Ad5 fiber protein, resulting in enhancement of adenovirus transduction. However, up until now no one has investigated whether and how cysteines influence the retargeting potential of Ad5RGD4C. We have previously shown that disulfide bond formation within Ad5NGR4C, whose HI loop comprises an aminopeptidase N-binding NGR motif that is flanked by 4 cysteines, is required for targeting properties of NGR4C. By simple pretreatment of purified viruses with the reducing agent dithiothreitol (DTT) and by genetic replacement of cysteines with glycines, we clearly showed that disulphide bonds are formed and that these bonds are important for receptor binding. In this study, by the same approach, we wished to investigate the influence of symmetrically situated cysteines on the retargeting potential of the RGD4C motif. In our experiments, we used a panel of different cancer cell lines that are poorly infected with wild type Ad5. To study the influence of disulphide bond formation, cells were transduced with Ad5wt (wild type Ad5), Ad5RGD4C (Ad5 with a CDCRGDCFC motif inserted in the HI loop of fiber protein) and Ad5RGD4G (Ad5 with a GDGRGDGFG motif inserted in the HI loop of fiber protein) as a control vector, all treated with DTT prior to measuring their transduction efficacy. While untreated Ad5RGD4C transduces cells much more efficiently than Ad5wt, the transduction efficacy of Ad5RGD4G is just slightly better than that of Ad5wt. Additionally, treatment with DTT dramatically decreased transduction efficacy of Ad5RGD4C on all examined cell lines, in a dose dependent manner, but had no influence on either Ad5RGD4G or Ad5wt. Based on these results, we conclude that the cysteines surrounding the RGD motif inserted in the HI loop of Ad5 form disulphide bonds that are important for its retargeting potential indicating that the cysteine-constrained RGD-peptide has an advantage over its linear counterparts. In the future we will investigate the relationship between expression patterns of RGD binding integrins on different cell lines and retargeting potential. We presume that different integrins could bind differentially to linear and cyclic RGD motifs.

adenovirus; fiber protein; disulphide bonds

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