Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks

Abstract

Construction of a complex virus may involve a hierarchy of assembly elements. Here, we report the structure of the whole human adenovirus virion at 3.6 angstroms resolution by cryo-electron microscopy (cryo-EM), revealing in situ atomic models of three minor capsid proteins (IIIa, VIII, and IX), extensions of the (penton base and hexon) major capsid proteins, and interactions within three protein-protein networks. One network is mediated by protein IIIa at the vertices, within group-of-six (GOS) tiles--a penton base and its five surrounding hexons. Another is mediated by ropes (protein IX) that lash hexons together to form group-of-nine (GON) tiles and bind GONs to GONs. The third, mediated by IIIa and VIII, binds each GOS to five surrounding GONs. Optimization of adenovirus for cancer and gene therapy could target these networks.

Figure 1

Overall structure of the Ad5 capsid. (A) Radially colored surface of a reconstruction of the capsid, centered on a 3-fold axis. (B) Views of the outer surface (upper) showing minor protein IX – and following rotation – the inner surface (lower) of a facet showing minor proteins IIIa, VI, and VIII. All hexons, penton bases, and penton fibers are shown semi-transparently except for one hexon monomer (+) and one penton base monomer (*). Upper left inset: Side view of protein IX among hexons. Lower right inset: Side view of proteins IIIa and VIII centered on a penton base. (C) Atomic model (sticks) of an α helix from a hexon monomer superimposed on its density map (mesh) with some side chains labeled.

Figure 2

Interactions among minor and major proteins on the inner surface. (A) Left: Global view of the arrangement of protein IIIa (red) and protein VIII (blue). Middle: Organization of hexon trimers into a GON (gray shade), peripentonal hexon trimers (light blue shade) and a penton-base pentamer (orange shade) into a GOS. Top insets: β augmentation at the VC regions of the H3 hexon trimer by the body (left) and the head (right) domains of protein VIII. Right insets: The upper inset shows β augmentation at the VIII-binding domain of protein IIIa by the body domain of protein VIII; the lower inset shows interactions among the N-arm of a penton base and two adjacent proteins IIIa. (B) Ribbon model of protein IIIa (aa 7-300) with four domains. (C) Ribbon model of protein VIII with three domains. Lower inset: Head domain density (semitransparent gray) and its atomic model (ribbon), showing cleavage sites G110 and R159 between the N-terminal portion (blue) and the C-terminal portion (red).

Figure 3

Interactions among minor and major proteins on the outer surface. (A) The physical network of protein IX on the outer surface lashes hexons together into GON tiles but avoids GOS tiles that are each centered on a vertex. Insets: Center left: Ribbon models of the N-terminal domains of three protein IX monomers (blue, green and red), overlying the models of three adjacent hexon (H2, H3, H4) monomers (gray) at a local 3-fold axis. Upper left insets: N-joint of three protein IX monomers and its side view, showing a hydrophobic core containing a triplet of tyrosines (Y14) and a triplet of leucines (L15). Lower left inset: β augmentation at the FG2 region of a hexon H2 by the N termimus of protein IX. Upper right: Four-helix bundle with three parallel and one anti-parallel α helices linked by a ladder of hydrophobic residues (leucines and valines, magenta). Lower right: Head-on view of the helix bundle and the hydrophobic core. (B) Ribbon model of protein IX with three domains and the N-joint region. (See also Figure S5.) Inset: Density map (mesh) and atomic model (sticks) of a representative loop from the N-terminal domain.

Figure 4

Newly resolved regions in penton-base and hexon proteins. (A) CryoEM model (ribbons) of the penton-base protein superimposed on its density map (semi-transparent gray). Outside the box, the cryoEM atomic model (red ribbons) is identical to the x-ray model (11). Inside the box is our newly resolved N-arm (blue ribbon, aa 37-51). Inset: Enlargement of the boxed region, showing side chain densities (mesh) and its atomic model (ribbon). (B) CryoEM model of the hexon protein. Red ribbons show agreement with the x-ray model (10). Blue ribbons show our newly resolved pieces, including the N-terminal and the C-terminal extensions. Region names in the hexon monomer (e.g., VC, FG, etc.) follow ref. (10). (C-D) Conformational adaptation. (C) Twelve hexon monomers exhibit five types of N-terminal extension in an asymmetric unit: four of type 1, two each of types 2 and 3, one of type 4, and three of type 5. (D) Twelve hexon monomers exhibit six types of C-terminal extension: two each of types a, b, c and d, three of type e, and one of type f. Ribbon models superimposed on density (mesh) of these six types are shown in Fig. S8A.

Figure 5

Schematic illustrations of interactions among minor and major proteins. Interactions are marked here, numbered in Fig. S10, and listed in table S3. (A) Contacts on the inner surface of the capsid. Characters a-f denote the positions of six types of hexon C-extensions. At each vertex, five copies of protein IIIa link five peripentonal hexon trimers and a penton-base pentamer to make a GOS tile (light blue shade). Protein VIII mediates binding among hexons, links GON tiles (gray shade) to GON tiles, and links GON tiles to GOS tiles. (B) Contacts on the outer surface among the four types of hexon trimer (H1, H2, H3, H4) and the four types of protein IX monomer (red, green, yellow, blue) that are inlaid into the canyons at the borders between hexons. Protein IX lashes hexons together to form GON tiles and also links GON tiles.