Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

Abstract

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

Structural Insights into Adeno-Associated Virus Serotype 5

“…Unlike the structures determined for some other AAV capsids, for example, AAV3B, AAV4, AAV6, and AAV8, including VLPs produced in baculovirus/Sf9 expression systems (e.g., AAV6 and AAV8), where at least one DNA nucleotide is ordered (22,23,26,27,29), there was no DNA ordered in the AAV5 capsid interior in the previously identified DNA binding pocket, despite the conservation of the binding pocket amino acids. The lack of ordering of a nucleotide in AAV5 is predicted to be due to the high radiation sensitivity of the thin crystals used for X-ray diffraction data collection.…”

“…An additional visual comparison of the superposition of the structures was carried out using the Coot program (62). The available crystal structures of AAV3b, AAV6, AAV8, and AAV9 (23,26,27,29) were not included in this analysis due to their high similarity to AAV2 and the fact that regions which vary between them and AAV2 were already described when this serotype was compared to AAV4.…”

“…Structures determined for AAVs show capsids assembled from the common VP3 region (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). There is currently no experimentally determined structure for VP1u or the overlapping VP1/ VP2 N terminus.…”

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

“…Structural Modeling-Coordinates for the AAV2 and AAV9 viral protein (VP) crystal structures were obtained from the RCSB Protein Data Bank (PDB codes 1LP3 and 3UX1, respectively) (14,15). Using the SWISS-MODEL protein structure modeling server (16), homology models of the 2G9 and 2i8G9 VP3 monomers were generated with crystal structures of AAV2 VP3 as template.…”

Functional Analysis of the Putative Integrin Recognition Motif on Adeno-associated Virus 9

“…Structural Modeling-Coordinates for the AAV2 and AAV9 viral protein (VP) crystal structures were obtained from the RCSB Protein Data Bank (PDB codes 1LP3 and 3UX1, respectively) (5,24). Three-dimensional models of the AAV2 and AAV9 VP3 subunit trimers were created using the Oligomer Generator utility in VIPERdb-Virus Particle ExploreR2 (25 …”