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.

Fig 1

Characterization and cryo-EM of baculovirus-expressed AAV9 VLPs. (A) SDS-PAGE of AAV9 showing VP1, VP2, and VP3, which have masses of 87, 73, and 62 kDa, respectively. (B and C) Micrographs of negatively stained (B) and vitrified (C) AAV9 VLPs. Size marker, 500 Å. (D) Resolution estimation using Fourier shell correlation (FSC) coefficient values and average phase difference between two half-data sets plotted versus spatial frequency for the temperature factor corrected (gray) and uncorrected (black) structures. The resolution was determined to be where the average phase difference increases to 50% or the correlation coefficient dropped below 0.5, indicated by the horizontal dashed line.

Fig 2

Comparison of AAV structures at a 9.7-Å resolution. (A) AAV9 cryo-EM density map. (B) Cross-section of the AAV9 cryo-EM density map with an octant removed to show the capsid interior. (C) A surface density map of the pseudoatomic model built into the AAV9 cryo-EM reconstructed density map. (D, E, and F) Surface density maps for AAV2, AAV4, and AAV8, respectively, generated from structure factors and phases calculated from atomic coordinates to a resolution of 9.7 Å. The capsid surfaces shown in panels A and C to F are radially depth cued with colors ramping from red, to yellow, to green, to cyan, and finally to dark blue. In panel B, the radial range for the depth cueing was expanded to enhance the internal surface features. All density maps are viewed down an icosahedral two-fold axis. The triangles in A and D depict a viral asymmetric unit bounded by a five-fold axis and two three-fold axes. Density for the DE and HI loops conserved in all parvovirus structures determined to date is indicated by arrows in A and C. Example capsid surface regions corresponding to VR-I and VR-IV are indicated by arrows in panels A and C to F. The images were generated using Chimera (59).

Fig 3

Modeling of the cryo-reconstructed AAV9 density map. (A) The AAV9 capsid pseudoatomic model (in blue) generated by SWISS MODEL (33) using the AAV2 and AAV8 crystal structures as templates. Two surface loops (VR-I) and (VR-IV) per capsid monomer required adjustment to fit within the cryo-EM density map (in gray mesh, contoured at 1.2 σ). The left half of the capsid is a cross-section to show the fit of the model in the capsid interior. (B) The AAV9 capsid crystal structure (in red) inside the cryo-EM map (in gray mesh; contoured at 1.2 σ). (C) The conserved α-helical region (αA) in the VP3 crystal structure shown inside the cryo-reconstructed density at a threshold of 3.8 σ. This region, which flanks the icosahedral two-fold axis in all parvovirus structures determined to date, was clearly resolved in the 9.7-Å-resolution cryo-reconstructed map. (D) Cross-section of an isosurface rendering (in gray) of the cryo-reconstruction overlaid with a difference map (F_o − F_c) calculated by subtracting the structure factors of the docked crystal structure from those of the cryo-EM map. Positive difference density is shown in green; negative difference density is shown in red. The positive tubular density is inside the five-fold channel. The images were generated using Chimera (59).

Fig 4

The AAV9 VP3 crystal structure. (A) AAV9 monomer indicating the conserved eight-stranded β-barrel motif present in all parvovirus capsids, as well as the conserved αA at the two-fold symmetry axis. Small regions of β-strand and α-helical structure are present in the loop regions between the β-barrel strands. The approximate positions of the icosahedral two-, three-, and five-fold axes are indicated with filled oval, triangle, and pentagon symbols, respectively. (B) Close-up view of VR-I and (C) VR-IV showing 2F_o − F_c density (contoured at a threshold of 1.5 σ) for the side chain atoms in these surface loops. The images were generated using PyMol (the PyMOL Molecular Graphics System version 1.3, Schrödinger, LLC.).

Fig 5

Structural alignment of AAV9 VP3 with the VPs of AAV2, AAV4, and AAV8. The VP3 regions from the crystal structures of AAV2, AAV4, AAV8, and AAV9 are compared. Identical residues are shown in white text and a black background, and nonconserved residues are in black with a white background. The eight β-strands that comprise the antiparallel β-barrel motif are depicted with open arrows, the conserved α-helix, αA, is indicated with an open rectangle, and the VR regions are labeled and identified with black arrows. Amino acids in VP regions that are not structurally equivalent to AAV9 are listed in an offset position, below the alignment for each serotype.

Fig 6

Superposition of AAV VP3 structures. (A) Overlay of the crystal structures of the VP3 monomers of AAV2 (blue), AAV4 (red), AAV8 (green), and AAV9 (brown), with locations of VR-I to VR-IX labeled. The eight-stranded β-barrel motif, DE loop, and HI loop are identified. The approximate locations of some icosahedral symmetry axes are indicated as in Fig. 4A. Panels B, C, and D show close-up views of the superposed structures of AAV2, AAV4, AAV8, and AAV9 for VR-I, VR-II, and VR-IV, respectively. The VR-I, VR-II, and VR-IV conformations as predicted by SWISS MODEL based on the crystal structures of AAV2 and AAV8 and as interactively modeled into the cryo-EM density map are shown in gray and black, respectively, in panels A to D. The images were generated using PyMol (the PyMOL Molecular Graphics System version 1.3, Schrödinger, LLC.).

Fig 7

Variable regions of the AAV capsid and associated functions. A trimer of AAV9 VP3 (in black, dark gray, and light gray) is shown viewed down an icosahedral three-fold axis (left) and at a slight rotation (right) with VR-I (brown), VR-IV (yellow), VR-V (lime), and VR-VIII (limon) highlighted. Residue positions equivalent to the heparan sulfate binding site for AAV2 (R484, R487, K532, R585, and R588) are shown in blue. The LamR receptor binding footprint for AAV8, residues equivalent to the amino acids in the two capsid regions aa 489 to 545 and 591 to 621 (AAV9 VP1 numbering), are shown in light and dark green, respectively. Residues 503 (orange) and 504 (purple) are part of the light green LamR footprint. Residues 591 to 594 (gray/green) in the dark green region of the LamR footprint are also part of VR-VIII.