Integrin alpha v beta 3 and disulfide bonds play important roles in NGR-retargeted adenovirus transduction efficiency

Aims: Adenoviruses that have CNGRCVSGCAGRC peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cys-teines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form di -sulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. Methods: Transduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13. Key findings: AdFNGR and AdHNGR attach better to CD13 and/or alpha v beta 3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of alpha v beta 3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on alpha v beta 3 integrin expression. Significance: Insertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors.