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

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

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

Tomograms of purified A/Udorn/72 virions (A).

<p>Three distinct morphologies were observed: short-rods (B), longer filaments (C) and spherical virions (D). Length and diameter measurements from 96 particles were plotted (E) showing that filamentous particles had a narrower diameter compared to the shorter rod-shaped particles (that we term bacilliform virions). Long filaments that extended beyond the field of view were plotted with a filled square. Spherical virion dimensions (those with an axial ratio <1.2) were plotted with a hollow circle. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s012" target="_blank">movie S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s006" target="_blank">figures S6</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s007" target="_blank">S7</a>.</p

RNP arrangement and other internal components in the various classes of virions.

<p>(A) Transverse sections through bacilliform particles revealed the characteristic arrangement of RNPs. (B) Longitudinal section of the particle in (A) showed three RNPs lying side-by-side. (C) Such views were commonly observed with particles oriented parallel to the ice layer and transverse sections through these particles (D) showed the 7+1 arrangement of RNPs. (E, F) Longer bacilliform particles had RNPs at one end while longer filaments (G) were sometimes sparsely packed but more frequently contained fibrillar material along their entire length (longitudinal and transverse sections are shown H–K). In some cases the internal density appeared as straight rods (H, I) while in others it appeared to be wound around itself (J, K). Tomogram sections perpendicular to the ice layer were harder to interpret owing to the missing wedge; an imaging artefact associated with tomographic data acquisition (B, D, I and K). (I and K) Sections through the narrow filaments do not show the RNP morphology seen in comparable views of bacilliform particles (A and D). This feature was infrequently seen at the termini of cell-associated filaments and in our data seen only once in tomograms of purified filaments (L). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s012" target="_blank">movie S5</a>.</p

Cryomicroscopy and tomography of influenza A/Udorn/72 infected cells.

<p>(A) Low magnification cryomicrograph of a long filament and Archetti body attached to a cell edge (red line). (B) A slice through a tomogram of the Archetti body shown in (A) reveals that the head was largely devoid of content. (C) Filaments over 10 µm long attached to a cell (red line). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s009" target="_blank">Movie S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s004" target="_blank">figures S4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s005" target="_blank">S5</a>.</p

Close up view of filament formation at 8 hours (A) and 10 hours (B) post-infection showing the presence of bulbous termini at the end of some long filaments (C, D) and (G, H) indicated with a yellow box.

<p>Many filaments were also seen that did not show stronger fluorescence at their termini, indicating that they were probably not Archetti bodies (E, F orange boxes). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s008" target="_blank">movie S1</a>.</p

Cell associated long filamentous structures.

<p>(A) Tomogram showing a large terminal varicosity and two filamentous particles that appear to contain RNPs at their ends (Insets 1 and 3). An extended helical structure is also seen, possibly M1 (Insets 1 and 2). (B, C) Archetti bodies and the majority of filamentous structures were seen not to have ordered arrangements of RNP at their ends. (D) A filamentous virion containing an extended helical structure that may be M1. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003413#ppat.1003413.s011" target="_blank">movie S4</a>.</p

Time course immunofluorescence imaging of filament formation in MDCK cells infected with Influenza A/Udorn/72.

<p>DAPI was used to stain cell nuclei (blue) while phalloidin was used to detect actin (red). Monoclonal antibodies were used to detect viral proteins; NP is shown in green and HA is shown in white. Budding virus is seen from as early as 6 hours post infection. From 8 hours we can see long filaments and Archetti bodies at the cell surface.</p