Crystal structure of human adenovirus at 3.5 A resolution

Abstract

Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here, we report the x-ray structure at 3.5 angstrom resolution of the 150-megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a substantial advance toward understanding the assembly and cell entry mechanisms of a large double-stranded DNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.

Figure 1

Surface rendering and subunit associations of Ad35F. (a) Outer capsid viewed down the particle 3-fold axis with a facet circumscribed by the white triangle. (b) A zoom in view of the single facet. The icosahedral asymmetric unit is composed of one of the PB (pink) subunits and 4 hexon trimers (cyan(1), red(2), green(3), yellow(4)). Cement proteins on the outside of the capsid are shown in blue (4-helix bundle) and magenta (IX triskelion). Twelve hexon trimers occupy one facet of the Ad35F capsid; however, a total of 18 hexons are shown for clarity. A model of the trimeric fiber docked into each penton base is shown in orange.

Figure 2

Structure of the penton complex. (a) The structure of the isolated PB shown in gold (10) with a maximum pore diameter of 28 Å. (b) The structure of the PB in the Ad35F particle shown in magenta with a maximum pore dimension of 50 Å. (c) Panel showing the 3-fold averaged difference (F_o−F_c) electron density (in blue, contoured at 2.5σ) corresponding to the fiber molecule along the 5-fold axis of the PB, shown as magenta trace. Structure of the HAdV-35 fiber molecule based on the partial HAdV-2 fiber structure (9), represented as ribbon diagram (red) and fitted into the difference density. (d) A vertical view of the fit of the fiber shaft to the difference density (left). The flexible 3^rd repeat of the shaft is indicated by an arrow. A view down the fiber axis showing the fit of one of the shaft repeats (right).

Figure 3

Hexon associations at the inner capsid surface. (a) Inner surface of a facet showing the locations of hexon C-termini, shown as spheres according to the hexon color-coding used in Fig. 1a. Group of nine (GON) hexons in the facet are shown in tan color, while the rest of the hexons are shown in gray for clarity. VIII molecules are shown in black. PB subunits are shown in pink and additional cement proteins in purple. Structurally distinct locations (A and B) of VIII molecules as well as the 4 hexon trimers (1, 2, 3 and 4) of the icosahedral asymmetric unit are indicated. Hexons related by icosahedral symmetry are indicated with the prime/double prime symbols. (b) Formation of extended β-sheets by the hexon C-termini (green) with the EF-strands (aa 721–730; purple) of the V2 domain of the neighboring (clockwise) hexon subunit at the icosahedral 3-fold axis (triangle). (c) Stacking of the hexon C-termini (red and green) at the inter-GON interface with a helix from VIII (black). Hexon N-termini that interact with VIII are shown in powder blue.

Figure 4

Structure and interactions of pVIII with the hexons. (a) Display of 2F_o−F_c electron density (contoured at 1.0 σ) of the ordered part of a VIII molecule. (b) An interior view of the facet showing the locations of proteins VIII (black) and additional cement proteins (purple). An outline of the group of nine hexons (GONs) is shown by a thick blue line. A line trace corresponding to the location of three P30 molecules (in white) from PRD1 (9) is shown in comparison with protein VIII and in the context of the Ad35F structure. Hexons are color coded and numbered according to the scheme described in Fig. 1a and Fig. 4a respectively. (c) Non-polar interactions between a VIII helix (black) with the N-termini (powder blue) of two neighboring hexons viewed from the inside of the virus. (d) Close approach of the inverted U-shaped bend in VIII (black) to the N-terminus of a hexon subunit. However, the interactions of the U-shaped structure are not as close and specific as those mediated by the helix in panel c.