Insights into the AAV packaging mechanism: Cryo-EM Structure of the AAV2 Rep-Capsid Packaging Complex.

The Adeno-associated virus (AAV) has become the most used viral vector for gene therapy applications to treat monogenic diseases, with over 250 clinical trials and six FDA-approved biologics. AAV has a single-stranded DNA genome encapsulated in a 60-subunit icosahedral protein shell. Assembly of empty capsids occurs in the nucleus, and in a subsequent step, the genome is packaged by the motor activity of the AAV Rep proteins. The translocation of ssDNA has been suggested to occur through one of the twelve channels at the fivefold symmetry axis. While there is substantial evidence that Rep proteins directly interact with the capsid, the specific molecular determinants, stoichiometry, and translocation mechanism remain unknown. To understand how Rep proteins assemble on the capsid, we examined Rep-capsid complexes using cryo-electron microscopy with single-particle reconstruction. Our results show that Rep proteins can assemble into the capsid fivefold pore as either pentameric or hexameric rings. The different ring complexes dock into the pore similarly, using the post-sensor1 β-hairpin motif (pos1βh2) as a capsid interaction module. This interaction induces the folding of a segment in the capsid HI loop, resulting in an expansion of the pos1βh2 β-sheet. Our structures show that any of the AAV Rep proteins can interact with the capsid, and this mode of interaction may be a conserved mechanism across all parvoviruses. Collectively, our results provide insights into the mechanism of AAV genome encapsidation and could inform strategies to improve recombinant AAV packaging efficiency.