DNA packaging in HSV-1.

<p>The unsharpened C5 reconstruction of HSV-1 is presented showing a series of radially cropped views of the interior density; these reveal the highly ordered arrangement of packaged DNA. The outermost (a), second (c), and third (e) shells are shown, revealing a left-handed spool of density. Spherical sections of each shell are also presented (b,d,f). HSV, Herpes Simplex Virus.</p

The structure of the portal-vertex interior.

<p>A central slice through the C5 reconstruction of the HSV-1 virion reveals the internal features of the portal-vertex (a). Notably, a strong linear density is seen to run through the portal-vertex that we attribute to genomic DNA (white arrow). The outermost feature, the PVAT, is weakly resolved as fuzzy density, suggesting that this feature is not well constrained. Isosurface representation of the unsharpened map presents a clearer representation of the PVAT (b), while in the sharpened density map, the packaged DNA is not seen, revealing the interior features of the capsid shell (c). A clipped, close-up view of the portal-vertex (boxed in c) highlights the morphology of the portal (pUL6) and, lying between the portal and the PVAT, the pentameric portal-vertex protein (wall-eyed stereo pair view, d). A close-up stereo view of the pentameric portal-vertex protein (boxed in d) clearly shows the density that is consistent with a two-helix coiled-coil motif (pink arrow, e). The density running through the centre of the portal-vertex that we attribute to DNA is also clearly visible (blue arrow). The density map was segmented to highlight three features: the portal (mauve), the pentameric portal-vertex protein (purple), and the periportal triplex–like density (magenta). The segmented portal-vertex is presented as stereo views both perpendicular to (e) and along (f) the portal axis. In panel e, the capsid and triplex-like assemblies are clipped to expose the pentameric portal-vertex protein; this and the portal are not clipped. In panel f, the pUL25/PVAT component is clipped away to expose the underlying features. HSV, Herpes Simplex Virus; PVAT, portal-vertex–associated tegument.</p

Stereo pair views of the structure and composition of the PVAT.

<p>Atomic coordinates for the CATC components pUL17, pUL25, and pUL36 (PDB 6CGR [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref005" target="_blank">5</a>]) and the C-terminal domain of pUL25 (PDB 2F5U [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref038" target="_blank">38</a>]) were docked into the portal-vertex density (a). Each pentonal five-helix CATC bundle has been shown to include two copies of an N-terminal α-helix of pUL25 (blue) along with two copies of a C-terminal α-helix of pUL36 (pink); these are bound to pUL17 (orange) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2006191#pbio.2006191.ref005" target="_blank">5</a>]. The atomic model of the penton-vertex CATC matches well the equivalent density at the portal-vertex, indicating that there are likely a total of 10 copies of pUL25 at the portal-vertex as well. The PVAT assembly comprises 10 globular densities arranged as two C5 symmetric star-shaped rings that crown the portal-vertex. We docked five copies of the atomic model for the C-terminal domain of pUL25 into the proximal (inner) tier (blue, b). The docked coordinates were then saved as a single model that was docked into the less well-defined distal tier (c). A side view of the CATC/PVAT components is shown (d). CATC, capsid-associated tegument complex; PVAT, portal-vertex–associated tegument.</p

CryoEM and 3D image reconstruction of HSV-1 virions.

<p>Views of unsharpened 3D reconstructions and close-up stereo pair images of the penton and portal vertices. Imposition of full icosahedral symmetry led to the calculation of a map at 6.3 angstroms resolution (a). At each 5-fold symmetry axis, the CATC binds to peripentonal triplexes to form an assembly that lays over the major capsid protein penton (white arrow, a). A sharpened map reveals higher-resolution features and in particular the CATC five-helix bundle (d,e). Focussed classification led to the calculation of a C5 symmetric reconstruction at 7.7 angstroms resolution (b). This revealed the structure of the unique portal-vertex (white arrow, b). In the sharpened map, the CATC five-helix bundle is also well resolved (g,h). A close-up view of the penton-vertex highlights the structure of the CATC—in particular, the two globular densities that have been attributed to the C-terminal domain of pUL25 (black arrows, c). The CATC five-helix bundle is highlighted by fitting of atomic coordinates for this assembly (extracted from PDB 6CGR) (boxed region, d); pUL17 is shown as an orange ribbon; two copies of pUL25 (N-terminal domains) are shown in shades of blue; and two copies of pUL36 (C-terminal domains) are shown in shades of pink (e). The Ta triplex is indicated by a pink arrow (e). A close-up view of the portal-vertex shows that the 5-fold symmetry axis is capped by a tiered structure comprising two star-shaped rings of density (the PVAT, f). The arms of the CATC are also angled more towards the 5-fold axis. The sharpened map, viewed at a higher threshold, does not show the distal tier of the PVAT (g). Rigid body docking of CATC coordinates gave a good fit to the density and revealed a 6° counter-clockwise rotation of this assembly compared to that of the penton-vertex (compare e and h). Interestingly, we see that the position occupied by the Ta triplex in the penton-vertex is occupied by a much larger globular density at the portal-vertex (pink arrow). All maps are coloured according to radius in angstroms (see colour key). CATC, capsid-associated tegument complex; cryoEM, electron cryomicroscopy; HSV, Herpes Simplex Virus; PVAT, portal-vertex–associated tegument.</p