Structure and uncoating of immature adenovirus

Abstract

Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

Fig 1

3DEM maps and quasi-atomic models. Central sections of the wild type (A) and ts1 (B) maps, both filtered at 8.9 Å resolution. The bar represents 200 Å. Higher density is white. (C) Resolution assessment. Fourier shell correlation curves for the wild type (wt) and ts1 3DEM maps. (D) Surface rendering showing the wild type AU, as seen from outside the virion. The 4 independent hexon trimers are labeled 1–4. Hexon 1 and its symmetry mates form the peripentonal ring; hexon trimers 2, 3 and 4 form the GONs. The bar represents 100 Å. (E) Ribbon representation showing the wild type quasi-atomic AU model. The four hexon trimers have been labeled as in (D) and depicted in different colors to facilitate interpretation. One penton base molecule is shown in dark blue. (F) A slab of the AU showing the good correspondence between the cryoEM density (semitransparent surface) and α-helices at the base of the hexon trimers, colored as in (E). Black filled symbols indicate the 5-fold (pentagon), 3-fold (triangle) and 2-fold (oval) icosahedral symmetry axes.

Fig 2

Differences between mature and immature capsids. (A) Details of sections (42 Å away from the virion center, looking along a 2-fold axis as in Fig. 1A and B) of the wild type (wt) and ts1 3DEM maps, as indicated. The positions of extra densities appearing in the ts1 map are indicated with a white square (type 1 difference, at the capsid-core interface) and circle (type 2 difference, inside the hexon cavity). (B) Surface rendering of the wild type-quasi atomic model (yellow) and ts1-wild type (red) difference maps superimposed on the quasi-atomic model density map (semitransparent). Note that the AU is shown as seen from inside the virus, i.e. rotated 180° around a horizontal axis with respect to Fig. 1D, E and F. The four independent hexon trimers and one penton base molecules are colored as in Fig. 1E. Black boxes and circles indicate the location of type 1 and type 2 difference densities. Black filled stars indicate the position of the second independent polypeptide VIII copy (the first one is located underneath the peripentonal ring). Black filled symbols indicate the 5-fold (pentagon), 3-fold (triangle) and 2-fold (oval) icosahedral symmetry axes. (C) Schematics showing an AU in the same orientation as in (B). The four hexon trimers are represented as hexagons, and the penton base as a pentagon. Black and grey shapes indicate the current model assignments for polypeptides IIIa, VI and VIII. Red boxes and circles indicate the location of the ts1-wild type type 1 and 2 differences. (D) A section across the icosahedral edge showing the inner cavities of hexon trimers 1 (green) and 2 (purple). External density in the wild type-quasi-atomic difference map (yellow) corresponds to the fiber (f) and hexon loops (hl). Colors and symbols as in (B). (E) Electrostatic surface coloring for a hexon trimer. The front half of the molecule has been clipped away to reveal charges within the cavity. All scale bars represent 50 Å.

Fig 3

Differences between mature and immature cores. (A) Radial average profile of the wild type and ts1 3DEM maps. (B) Examples of disrupted virions found in cryo-EM preparations of wild type (wt) and ts1 samples, as indicated. An arrow indicates an intact particle. The scale bar represents 100 nm.

Fig 4

Disassembly assays. (A) Analysis of DNA release for wild type and ts1 virus measured by extrinsic PI fluorescence at 607 nm as a function of temperature. Average values and error bars indicating standard deviations for triplicate measurements are plotted. (B) Negative stain electron microscopy images of wild type and ts1 disassembly intermediates obtained at 45 or 47°C, as indicated. The scale bar represents 200 nm.