Structural insights into adeno-associated virus serotype 5

Abstract

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.

Fig 1

Structure of AAV5. (A) A section of the averaged 2F_o − F_c electron density map (gray mesh, contoured at a threshold of 1.0 σ) for residues 385 to 389 shown in stick form and colored according to atom type: C in yellow, N in blue, O in red, and S in green. (B) A ribbon diagram representation of the ordered overlapping VP3 monomer region (residues 209 to 724) of AAV5. The conserved β-barrel core motif (βBIDG-βCHEF, gray), conserved αA helix, DE loop (between βD and βE), HI loop (between βH and βI), and subloops between βG and βH (GH-L1 to GH-L5) are labeled. The approximate positions of the 2-, 3-, and 5-fold axes are indicated by the filled oval, triangle, and pentagon, respectively. The interior and exterior capsid surfaces are also indicated. The dashed red circle delineates the subloops within the GH loop. These images were generated using the PyMol program (http://www.pymol.org).

Fig 2

AAV capsid surfaces. (A, C, and E) Exterior capsid surfaces for AAV5, AAV2, and AAV4, respectively. The surfaces are radially color cued (from capsid center to surface: blue to green to red to yellow; ∼80 to 140 Å). The white triangles depict the viral asymmetric units bounded by a 5-fold (5f) axis and two 3-fold (3f) axes divided by a line through a 2-fold (2f) axis. Example surface features that differ between AAV5 and the AAV2 and AAV4 compared are indicated: VR-IV, VR-VII, DE loop, and HI loop. (B, D, and F) Cross-sections of the AAV5, AAV2, and AAV4 capsids, respectively, showing their interior surfaces. The radial depth cue is as described for panels A, C, and E. The dark blue regions show the βA strand and βBIDG-sheet secondary-structure elements conserved in the three viruses. The 2-fold (2f) axis is indicated in AAV5. These images were generated using the CHIMERA program (118).

Fig 3

Unique features of the AAV5 VP3. (A) Superposition of the AAV2 (blue), AAV4 (red), and AAV5 (gray) VP3 monomer structures shown in a coil representation. A B factor (temperature factor) putty (thicker-coil) representation is used to show the regions of highest variability. The DE loop, HI loop, VR-I to VR-IX, and N and C termini are labeled. The approximate icosahedral symmetry axes are indicated as described in the legend of Fig. 1. (B) Structure-based sequence alignment of AAV2 (blue), AAV4 (red), and AAV5 (gray) for the amino acids in VR-IV, VR-V, VR-VI, VR-VII, and the HI loop. Regions of structural differences to AAV2 are offset above (for AAV5) or below (for AAV4) the alignment. (C) Superposition of the AAV2 (blue), AAV4 (red), and AAV5 (gray) VP3 structures at VR-V, VR-VI, and VR-VII (coil representation) within the averaged 2F_o − F_c electron density map of AAV5 (gray mesh) contoured at a threshold of 1.0 σ. (D) Superposition of the HI loop (coil representation) of the VP3 structures of AAV2 (blue), AAV4 (red), and AAV5 (gray) within the averaged 2F_o − F_c electron density map of AAV5 (gray mesh) contoured at a threshold of 1.0 σ. In panels C and D, amino acid side chains have been omitted for clarity. (E and F) Positions of VR-I to VR-IX within a viral asymmetric unit (defined in the legend of Fig. 2). The viral asymmetric unit contains contributions from the reference VP3 monomer and a 2-fold (2f), 3-fold (3f1), and 5-fold (5f1) (see reference for definition of symmetry relationships) VP3 monomer (Fig. 1B). The prefixes on the labels for the variable regions indicate the contributing monomers. Panels A, C, and D were generated using the PyMol program (http://www.pymol.org), and panels E and F were generated using the Bobscript program (119) and rendered with the RASTER3D program (120, 121).

Fig 4

AAV5 capsid interactions with glycerol and cations. (A) A coil representation of the AAV5 VP3 (gray) with the HI loop (stick representation) from a 5-fold-symmetry-related VP3 monomer (wheat). The ordered glycerol molecule and three Na⁺ ions are shown in the averaged AAV5 2F_o − F_c electron density map (gray mesh) contoured at a threshold of 1.0 σ. The side chains for the amino acids interacting with the glycerol and Na⁺ ions are shown in stick representation colored according to atom type (C in green, N in blue, and O in red) and labeled. The HI loop and VR-I to VR-IX are labeled. The approximate icosahedral symmetry axes are indicated as described in the legend of Fig. 1. (B). Stereo view of the averaged 2F_o − F_c electron density map (gray mesh, 1.0 σ threshold) for the ordered glycerol molecule and its surrounding amino acids shown in stick presentation and colored as described for panel A for the reference VP3 monomer: C in cyan, N in blue, and O in red for the 5-fold-symmetry-related VP3 monomer; C in yellow and O in red for the glycerol. Distances (in Å) between the glycerol and interacting amino acids are indicated. The images were generated using the PyMol program (http://www.pymol.org).

Fig 5

Reported determinants of AAV5 capsid assembly, receptor recognition, and transduction. An exterior capsid surface representation for an AAV5 VP3 trimer in gray (Ref, reference), green (3f1, 3-fold), and salmon (3f2, 3-fold). Surface regions of the VP3 monomers with the amino acid deletions (T492 to N493, red) or duplications (T492, red; Q574 to S575, blue) investigated by Hida et al. (113) are indicated by arrows and labeled. Residue A581, which when mutated to T affects AAV5 sialic acid binding by AAV5 and capsid internalization (102, 103), is shown in pink and labeled. VR-IV, VR-V, and VR-VIII, which interact from 3-fold-symmetry VP3 monomers to assemble the protrusions surrounding this axis (3F), are indicated by the dashed arrows with the open heads. This image was generated using the CHIMERA program (118).

Fig 6

A model of AAV-Go.1 based on the AAV5 VP3 structure. (A) A surface representation of six VP3 monomers, including the reference (Ref) monomer in gray, of the AAV-Go.1 homology model. Residues differing between AAV5 and AAV-Go.1 and occurring on the capsid surface are shown in purple, indicated with arrows, and labeled. These cluster on the 3-fold protrusions and the raised capsid region between the depression at the 2-fold axis and surrounding the 5-fold axis. The light gray triangle depicts the viral asymmetric unit as defined in the legend of Fig. 2. (B). A Roadmap (68) representation of the AAV-Go.1 model showing the capsid surface positions of the residues that differ with respect to AAV5 in the context of a viral asymmetric unit (black triangle). The residues that differ between AAV5 and AAV-Go.1 are shown in orange. Panel A was generated with the PyMol program (http://www.pymol.org), and panel B was generated with the RIVEM program (68).