AAV2 bypasses direct endosomal escape by using AAVR to access the trans-Golgi network en route to the nucleus

Vectors based on the adeno-associated virus are widely used as delivery methods in gene therapy applications, yet understanding of the mechanisms governing its intracellular trafficking remains incomplete. Traditional models suggest that AAV escapes from endosomes via membrane disruption, but direct evidence for this process is lacking. Here, we show that AAVR, the essential AAV cell entry receptor, functions as a bona fide retromer cargo. Using in vitro reconstitution assays, we demonstrate that AAVR’s cytosolic tail is sufficient to engage the retromer–SNX3 complex and drive membrane tubulation, a hallmark of retrograde trafficking. In AAVR-knockout HuH-7 cells, AAV2 particles are internalized, but accumulate in early endosomal compartments, and fail to support transgene expression. Galectin-8 recruitment assays reveal no evidence of endosomal membrane rupture during productive transduction, distinguishing AAV2 from lytic vectors such as lipid nanoparticles. Moreover, VP1u-deficient and PLA2-mutant AAV2 capsids accumulate at the TGN, indicating that VP1u is dispensable for early trafficking but required for post-TGN progression toward productive transduction. These findings argue against the widely held model of direct endosomal escape and provide a mechanistic basis for the previously described essential role of the TGN in productive AAV transduction, positioning AAV entry as a vesicle-guided, receptor-mediated process. IMPORTANCE The study demonstrates that AAV2 does not escape endosomes via membrane rupture but instead exploits the cellular receptor AAVR as a retromer cargo to traffic through vesicle-mediated retrograde transport to the trans-Golgi network, a critical checkpoint for productive transduction. Building on prior observations that VP1u-deficient particles are still internalized (R. Popa-Wagner, M. Porwal, M. Kann, M. Reuss, et al., J Virol 86:9163–9174, 2012, https://doi.org/10.1128/jvi.00282-12 ), we show that these particles traffic to the TGN but accumulate at this compartment, indicating that VP1u and its PLA2 activity become essential specifically at a post-TGN step in the transduction pathway.