AAV2-CAG-Cre-2A-GFP

Cellular entry of all serotypes appears to occur via receptor-mediated endocytosis. Single virus tracking techniques revealed that AAV2 virions typically contact the cell membrane multiple times before internalization occurs. This behavior most likely reflects the interaction of the capsid with heparan sulfate proteoglycans (HSPGs), the primary receptors for AAV-2. Binding to HSPGs may induce a reversible structural rearrangement of the capsid that is required for the next step of viral entry, which is dependent on coreceptors. Five coreceptors have been proposed so far. Fibroblast growth factor receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and laminin receptor are likely to enhance the virus:cell contact, facilitating thereby the HSPG induced structural rearrangement of the capsid, whereas integrins (αvβ5, α5β1) are thought to mediate the endocytosis of AAV-2. Integrins may also facilitate the trafficking of AAV from the periphery of the cell towards the perinuclear area by inducing cytoskeletal rearrangements. Viral particles appear to move inside endosomes. Endosomal acidification triggers conformational changes in the viral capsid, resulting in exposure of previously hidden regions, such as the N-terminus of VP1 containing a phospholipase A2 homology domain. This domain is conserved among parvoviruses and its function is required for AAV-2, most likely for endosomal escape. Once AAV capsids are released from endosomes, ubiquitination occurs, marking the capsid for proteasomal degradation. Consistent with this, addition of proteasome inhibitors results in increased transgene expression from AAV-based vectors. In addition to inhibiting AAV degradation, proteasome inhibitors may also affect translocation of the viral genome into or accumulation within the nucleus.