engleski

NGR-bearing adenovirus type 5 vectors use lipid rafts for cell entry

Human adenoviruses are non enveloped dsDNA viruses currently divided into 58 serotypes, adenovirus type 5 (AdV5) being one of the most studied. AdV5 infection starts with binding of fiber protein to coxsackievirus and adenovirus receptor (CAR), followed by internalization of viral particle via binding of an exposed RGD motif on the penton base to v integrins. Wild-type AdV5 internalization is mostly mediated by clathrin-dependent receptor- mediated endocytosis. Once in the cell cytoplasm, virions travel along the microtubules toward the nucleus where they subsequently dock to the nuclear pore complex and release their DNA into the nucleus. The efficiency of wild-type AdV5 infection in vitro is mainly determined by CAR expression. In vivo, CAR but also blood factors and integrins were shown to play a role in cell transduction. However, tumor cells very often do not express CAR or express this receptor at low levels. Capsid genetic engineering was used to retarget AdV5 toward new receptors/molecules specifically expressed on target cells/tissues. Previously, we have constructed several AdV5 retargeted towards aminopeptidase N (APN) by incorporating NGR peptide either in AdV5 fiber or hexon protein (AdV5-NGR) (Majhen et al. Biochem. Biophys. Res. Commun. 2006, 348:278-287 ; Jullienne et al. Gene Ther. 2009, 16:1405-1415). The present study aims at definying more precisely endocytic pathways used by AdV5-NGR. Here we showed that inhibiting clathrin-mediated endocytosis by ?? in CAR-negative APN-positive cells decreased transduction efficacy of wild-type AdV5, but the impact on AdV5-NGR transduction was much less pronounced. Additionally, by means of confocal microscopy, we observed the co- localization of wild-type AdV5 and transferrin (marker of clathrin-mediated endocytosis) in the early phase of internalization, and the co- localization between AdV5-NGR and transferrin in the late stage of internalization. This result suggested that AdV5-NGR can use some additional pathway/s besides clathrin-mediated endocytosis for cell entry. To test our hypothesis we analyzed the role of lipid raft-mediated endocytosis in AdV5-NGR transduction. Inhibition of lipid rafts by ?? in a CAR-negative APN-positive cell line significantly diminished transduction efficacy of AdV5-NGR, whereas it had almost no influence on cell transduction by wild-type AdV5. Moreover, we observed a strong co-localization between AdV5-NGR and cholera toxin B subunit (CTB, marker of lipid rafts) at both binding and internalisation steps of virus entry. In contrast, no co-localization between wild-type AdV5 and CTB was observed. Altogether, our results point out the important role of lipid raft-mediated endocytosis in AdV5- NGR transduction of CAR-negative APN-positive cells. To the best of our knowledge, this is the first study describing endocytic pathways used by AdV5 modified by incorporation of targeting ligands into hexon protein.

adenovirus; lipid rafts; NGR; aminopeptidase N

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