Functional analysis of the putative integrin recognition motif on adeno-associated virus 9 [Cell Biology]

November 17th, 2014 by Shen, S., Berry, G. E., Castellanos Rivera, R. M., Cheung, R. Y., Troupes, A. N., Brown, S. M., Kafri, T., Asokan, A.

Adeno-associated viruses (AAV) display a highly conserved Asn-Gly-Arg (NGR) motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In the current study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and non-specific sequestration by the spleen. Transient vascular hyper-permeability, induced by histamine co-injection exacerbated AAV9/NGA uptake by the spleen, but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding, but a more pronounced defect in cellular entry of mutant virions. These findings are corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level as well as systemic clearance by the reticulo-endothelial system.