We investigated the transduction of HEK293T cells permissive to adeno-associated virus serotype 8 (AAV8) to understand the mechanisms underlying its endocytic processing. Results showed that AAV8 enters cells through clathrin-mediated endocytosis followed by trafficking through various endosomal compartments. Interestingly, compared to the relatively well-characterized AAV2, a distinct involvement of late endosomes was observed for AAV8 trafficking within the target cell. AAV8 particles were also shown to exploit the cytoskeleton network to facilitate their transport within cells. Moreover, the cellular factors involved during endosomal escape were examined by an in vitro membrane permeabilization assay. Our data demonstrated that an acidic endosomal environment was required for AAV2 penetration through endosomal membranes and that the cellular endoprotease furin could promote AAV2 escape from the early endosomes. In contrast, these factors were not sufficient for AAV8 penetration through endosomal membranes. We further found that the ubiquitin-proteasome system is likely involved in the intracellular transport of AAV8 to nucleus. Taken together, our data have shed some light on the intracellular trafficking pathways of AAV8, which, in turn, could provide insight for potentializing AAV-mediated gene delivery.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health (R01AI68978 and P01CA132681) and a translational acceleration grant from the Joint Center for Translational Medicine.