7. Adeno-Associated Virus Type 2 (AAV2) Contains an Integrin alpha5beta1 Recognition Sequence Essential for Viral Cell Entry

Abstract

Integrins have been implicated as co-receptors in the infectious pathways of several non-enveloped viruses. For example, adenoviruses are known to interact with several alphaV integrins by virtue of a high affinity arginine-glycine-aspartate (RGD) domain present in the penton base of the capsid. In case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alpha Vbeta5 has been identified as a co-receptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as co-receptors by AAV2 have not been determined thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative co-receptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions are mediated by a highly conserved asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. Mutation of this domain reduces transduction efficiency by 10-fold ompared with wild type AAV2 in vitro and in vivo. Further characterization of mutant and wild type AAV2 capsids using cell surface and solid phase binding assays, peptide inhibition studies, and transduction assays in cell lines lacking specific integrins confirmed the role of the NGR motif in promoting AAV2-integrin interactions. Molecular modeling studies suggest that the NGR domain forms a surface loop located at the three-fold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. Based on the aforementioned results, we propose that cellular internalization of AAV2 follows a |[ldquo]|click-to-fit|[rdquo]| mechanism that involves co-operative binding of heparan sulfate and alpha5beta1 integrin receptors by the AAV2 capsid.