Integrin αvβ3 and disulfide bonds play important roles in NGR-retargeted adenovirus transduction efficiency

• Published in: Life sciences (1973) 2022-02, Vol.291, p.120116-120116, Article 120116
• Main Authors: Nestić, Davor, Hozić, Amela, Brkljača, Zlatko, Butorac, Ana, Pažur, Kristijan, Jullienne, Betsy, Cindrić, Mario, Ambriović-Ristov, Andreja, Benihoud, Karim, Majhen, Dragomira
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Adenoviridae - metabolism; Adenovirus vector; Adenoviruses; CD13; CD13 antigen; Cell Line, Tumor; Chemical bonds; Disulfide bonds; Disulfides - chemistry; Dithiothreitol; Efficiency; Endothelial cells; Endothelial Cells - metabolism; Expression vectors; Flow cytometry; Genetic Vectors - genetics; HEK293 Cells; Humans; Insertion; Integrin; Integrin alphaVbeta3 - metabolism; Integrin alphaVbeta3 - physiology; Integrins; Internalization; Mass spectrometry; Mass spectroscopy; Molecular modelling; NGR retargeting peptide; Oligopeptides - pharmacology; Peptides; Proteins; siRNA; Transduction; Transduction, Genetic - methods; Transfection; Transfection - methods; Viruses
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/j.lfs.2021.120116

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 cysteines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. 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. AdFNGR and AdHNGR attach better to CD13 and/or αvβ3 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 αvβ3 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 αvβ3 integrin expression. 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. [Display omitted] •AdFNGR and AdHNGR bind better to CD13 and/or αvβ3 integrin-positive cells than Adwt.•Reducing disulfide bonds by using dithiothreitol decreased transduction efficiency and binding of both AdFNGR and AdHNGR.•Mass spectrometry analysis indicate that cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds.•Retargeting properties of AdFNGR and AdHNGR rely mainly on αvβ3 integrin expression.