Differential role of αvβ3 and αvβ5 integrins in internalization and transduction efficacies of wild type and RGD4C fiber-modified adenoviruses

In order to analyze the role of αvβ3 and αvβ5 integrins in gene transfer by adenovirus-based vectors, an RGD4C motif was inserted into the HI-loop of wild type or shortened fiber protein of human adenovirus of serotype 5, thereby creating Ad5RGD4C and Ad5Δ639RGD4C vectors, respectively. Infection by the latter is independent of the Coxsackie B adenovirus receptor. Internalization and transduction of these vectors and were investigated in several stably transfected cell clones derived from a human laryngeal carcinoma cell line (HEp2) expressing different ratios of αvβ5 and αvβ3 integrins. We show that αvβ3 is more successful than αvβ5 in: (i) mediating adenovirus internalization and transduction when the RGD motif is present only in the penton base and (ii) mediating internalization and transduction by RGD4C-fiber modified adenoviruses. The highest amount of internalized virus was found in the cell clone in which αvβ3 integrin predominated over αvβ5 integrin (as judged by the % of cells expressing αvβ3 and αvβ5 integrins). However the level of transgene expression in this cell line was even lower than that in parental HEp2 cells which do not express αvβ3 integrin. This discrepancy between internalization and transgene expression (transduction) is likely due to the crucial role of αvβ5 in membrane permeabilization, indicating that αvβ5 integrin is a limiting factor for Ad5-mediated gene transfer. We conclude that αvβ3 integrin is an efficient adenovirus internalization receptor, but cannot functionally replace αvβ5 in endosomal release.