AAV9 Virus-Like Particles (Empty Capsids)

There are 13 different adeno-associated virus (AAV) serotypes that are structurally homologous and similar in their capsid proteins (VP1 to -3) in amino acid sequence. However, AAV9 is one of the most commonly studied vectors for use as a gene therapy vector. This is in part due to the ability of AAV9 to cross the blood-brain barrier and readily transduce a variety of tissues, including the central nervous system. Here, researchers report on the structures of AAV9 capsids determined at endosomal pH (7.4, 6.0, 5.5, and 4.0) using cryo-electron microscopy and three-dimensional image reconstruction, as well as AAV9 capsids with terminal galactose attached at pH 7.4 and 5.5. They observed for the first time that acidification induces externalization of the VP3 and possibly VP2 N termini, which may be a prelude to externalization of VP1u at pH 4.0, which is critical for lysosomal membrane disruption. Furthermore, structural studies of the AAV9-galactose interaction revealed that AAV9 remains attached to its glycan receptor at pH 5.5 in late endosomes. This interaction significantly alters the conformational stability of the VP variable region I, as well as the kinetics associated with the externalization of the VP N terminus.

WT AAV9 virus-like particles (VLPs) were purified and dialyzed to four different pHs, mimicking those encountered during endosomal maturation. AAV9 VLPs of each pH stage remained intact and, consequently, could be vitrified and subjected to data collection and structure determination by cryoEM. Researchers have obtained near-atomic, high-resolution 3D structures of the WT AAV9 capsid at the four pHs examined, with resolutions of 2.8 (pH 7.4, PDB ID 7MT0), 2.7 (pH 6.0, PDB ID 7MTG), 2.8 (pH 5.5, PDB ID 7MTP), and 3.0 Å (pH 4.0, PDB ID 7MTW), respectively (Figure 1).

Figure 1. Structural studies of the AAV9 virus-like particles (VLPs) at four different pHs, representing the acidification stages of endosome maturation. (Penzes J J, et al., 2021)