Latest insights on adenovirus structure and assembly

Abstract

Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

Keywords: adenovirus; core proteins; cryo-electron microscopy; crystallography; maturation; minor coat proteins; structure.

Figure 1

Overall AdV structure and components. (a) Icosahedral shell organization according to current structural knowledge. The left hand side panel is a model built from a low resolution cryoEM map, with penton bases highlighted in yellow, and fibers built from the crystal structure of the knob and distal shaft [27] in dark blue. The shaded triangle indicates one facet. (b) Non-icosahedral components. A segment has been removed from the cryoEM map to show the inner capsid contents. The schematics on the right hand side indicate tentative positions, as little is known about the structure and organization in the virion of the genome and accompanying proteins. Polypeptide IVa2, which binds to the specific packaging sequence in the viral genome, has been reported to occupy a singular vertex in the capsid [28].

Figure 2

Geometrical landmarks in the AdV icosahedral net. (a) Each hexon trimer is depicted as a hexagon with an overlaid triangle indicating the position of the towers. The two different kinds of faces in the hexon pseudo-hexagonal base are labeled S (single monomer) and T (two monomers). (b) One penton and the five adjacent facets are represented, spread out for clarity. The four hexon trimers forming the asymmetric unit (AU) are numbered in one facet. Peripentonal hexons, forming the Group of Six (GOS) together with the penton, are highlighted in yellow. Hexons in the Groups of Nine (GON) are depicted in white for the reference facet, gray in the rest. Red symbols indicate the icosahedral symmetry axes. Notice that there is an alternative way to define the AU, in which hexons belonging to two facets would be included (c). This alternative AU was used in the atomic resolution cryoEM study.

Figure 3

Organization of the AdV vertex region (GOS). (a) View from inside the capsid looking at the 5-fold icosahedral symmetry axis. (b) Side view showing a transversal section across the penton. The five peripentonal hexons are shown in gray; penton base in blue; polypeptide IIIa in yellow; and the GOS copy of polypeptide VIII in tan. Surfaces were created from high resolution structures in PDB ID 3IYN [4], using UCSF Chimera software [113]. Notice the N-terminal penton base arms (arrows) intercalating between IIIa monomers, and the radial position of polypeptide VIII, wedged between IIIa and hexon.

Figure 4

Functional motifs of polypeptide VI. The adenoviral protease (AVP) cleavage sites (scissor symbol) and the position of some residues are indicated below the gray bar representing the precursor polypeptide VI polypeptide chain.

Figure 5

Cementing of the GON. Organization of minor coat proteins VIII and IX in the GON. The four copies of IX in one GON are shown in blue, and the three copies of VIII in yellow. Only the fragments of IX that could be traced in the atomic resolution cryoEM map are depicted. Hexon positions are represented as transparent hexagons to allow simultaneous view of IX and VIII. Hexons in one AU are labeled 2 to 4 (hexon 1 is not a part of the GON). The view is from outside the capsid.

Figure 6

Polypeptide IX structure and conservation. (a) The extended fold of polypeptide IX is shown as a ribbon, with the different domains labeled and depicted in different colors. (b) Alignment of representative polypeptide IX sequences for the different species in the Mastadenovirus genus. In the bottom line the extent of the N-terminal (N), rope (r) and helix bundle (H) domains from the HAdV-5 structure are indicated with letters in the same color used for each domain in (a). Notice that the rope domain is conserved only in human and simian viruses (sequences within a black rectangle). Sequences were retrieved from UniProt [143] and aligned with Clustal [144] and JalView [145]. The extra-long PAdV-3 IX sequence is shown truncated for figure clarity.

Figure 7

Virion components undergoing proteolytic maturation. Each HAdV-5 precursor protein is represented as a bar with the polypeptide length in amino acids indicated in the center. Cleavage sites are denoted by arrows. There are four potential cleavage sites in pTP but they have not been experimentally verified. The prefix “p” denotes the unprocessed precursors.

Figure 8

The molecular stitch in immature AdV. (a) Schematics showing four adjacent icosahedral facets, with the position for the molecular stitch inside the capsid in red. (b) The GOS represented as in Figure 5, with the density for the molecular stitch derived from the ts1-WT difference map at 8.9 Å in red. The five peripentonal hexons are shown in gray; penton base in blue; polypeptide IIIa in yellow; and the GOS copy of polypeptide VIII in tan. View from inside the capsid looking at the 5-fold icosahedral symmetry axis. (c) Zoom into the region close to one of the molecular stitches with the structures of IIIa and VIII from [4] represented in ribbons. (d) Bars representing the precursor polypeptides IIIa and VIII with the cleavage sites indicated (arrows). Polypeptide regions not traced in the cryoEM atomic structure are in gray. Untraced regions close to the molecular stitch are indicated with the same symbol in (c) and (d).