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

NS4 expression profile.

<p>Confocal microscopy of BFAE cells infected with BTV-1 at a MOI of 1.5. Cells were fixed before infection (0 h) and at 0 h30, 2 h, 4 h, 8 h, 16 h and 24 h post-infection and processed for immunofluorescence using antibodies against VP7, NS1, NS2, NS3 and NS4 with an Alexa Fluor 488 secondary antibody as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a>. Scale bars correspond to 21.16 µm for 0 h to 4 h post-infection panels, and 13.6 µm for 8 h to 24 h post-infection panels.</p

The NS4 of BTV-1 displays similar biological properties to the homologous BTV-8 protein.

<p>(A) Western blotting of cellular extracts (lysate) of CPT-Tert cells infected with BTV-8 wt or BTV-8ΔNS4 at a MOI of 0.01. Cells were analyzed 24 h post-infection and blots were incubated with antisera against NS1, VP7, VP6, NS4 and γ-tubulin as indicated. (B) Schematic diagram of the BTV-8/BTV-1 reassortants and mutants used in this study. Note that BTV1 and BTV8 segments/proteins are coloured in blue and red, respectively. * indicates a point mutation, while # indicates the introduction of a stop codon in the NS4 ORF. (C) CPT-Tert cells were treated with 1000 AVU/ml of Universal IFN for 20 h prior, and 2 h after, being infected by the recombinant viruses indicated in the panel using a MOI of 0.01. Cell monolayers were stained 72 h post-infection using crystal violet. Values indicated below each well correspond to the relative quantification of the disrupted monolayer using Image-Pro Plus (MediaCybernetics, Inc.). (D) CPT-Tert cells were treated (solid line) or mock treated (dashed line) with 100 AVU/ml of Universal interferon (UIFN) for 20 h prior and 2 h after being infected by the viruses indicated in the panel. Cells were infected at a MOI of 0.01. Supernatants were collected at 24, 48 and 72 h after infection, and then titrated on BSR cells by limiting dilution analysis and virus titers expressed as log₁₀ (TCID₅₀/ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed two times, each time in duplicate.</p

BTV expresses a fourth non structural protein (NS4).

<p>(A) BTV segment 9 (1049 base pairs). The VP6 protein (dark gray) is encoded by nucleotides 16 to 1002. The NS4 coding sequence is located in the +1 open reading frame (ORF) between nucleotides 182 to 418. VP6 (residues 57 to 135) and NS4 (residues 1 to 79) amino acid conservation plots are shown. NS4 secondary structure prediction indicated the presence of two putative α-helices, drawn in blue and red. The N-terminal domain (blue) is highly basic and the C-terminal domain (red) contains a conserved leucine zipper motif. (B) Western blotting of cellular extracts (lysate) of BSR cells either transfected with 1.8 µg of plasmid expressing NS4 alone (pcI-NS4) or in fusion with eGFP (peGFP-NS4), or infected by BTV-8 or BTV-1 at a MOI of 0.01. Cells were analyzed 36 h post-transfection or infection and blots were incubated with NS4 antiserum. (C) Western blots of viral pellets and cell protein extracts of BFAE cells infected by BTV-1 at a MOI of 0.05. Samples were analyzed at 48 h post-infection by SDS-PAGE and western blotting employing antisera against NS1, VP6, VP7, ORFX (NS4) and γ-tubulin as indicated. (D) Immunohistochemical detection of NS4. Immunohistochemistry was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a> in brain tissue sections of mice inoculated with BTV-8 72 h post-infection using an antiserum against NS4. Cells expressing NS4 are stained brown as indicated by white arrows. No expression of NS4 is detected in negative control mice mock-inoculated with cell culture media.</p

Generation of ΔNS4 Bluetongue viruses by reverse genetics.

<p>(A) BTV segment 9 open reading frames. VP6 amino acid residues are written in black, NS4 amino acid residues are written in grey. The nucleotides at positions 183 (T), 252 (G) and 381 (T) were mutated to C, A and A, respectively (bold). Note that whilst these mutations do not change any amino acid residues of VP6, they remove the initiation codon of NS4 (position182) and introduce two stop codons into the NS4 coding sequence at amino acid positions 24 and 67. (B) Transfected BSR cells with BTV transcripts generated <i>in vitro</i> (0.5×10¹¹ molecules per segment for BTV-1 and 1×10¹¹ molecules per segment for BTV-8). Cell monolayers were stained using crystal violet at 72 h post-transfection. As negative controls, ΔVP6 assays correspond to using a segment 9 containing a stop codon at position 79 in the VP6 gene. (C) Agarose gel (1.5%) of purified BTV genomic dsRNA. BSR cells infected at a MOI of 0.01 were collected at 72 h post infection and BTV dsRNA was purified as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a>. 2 µg of dsRNA was loaded in each lane. (D) Western blotting of cellular extracts (lysate) of BSR cells infected at a MOI of 0.01. Cells were analyzed 36 h post-infection and blots were incubated with antisera against VP7, NS4 and γ-tubulin as indicated. Note that the double NS4 band in the BTV-1 sample is not a feature observed consistently. (E) Electron microscopy of BSR cells infected by BTV1-ΔNS4. Note cells display all the major features of BTV-infected cells including NS1 tubules (T), viral inclusion bodies (VIB) and viral particles (arrows). Scale bar = 1 µm.</p

<i>In vitro</i> growth properties of rescued WT and ΔNS4 viruses during interferon treatment.

<p>CPT-Tert cells were treated (solid line) or mock treated (dashed line) with 1000 AVU/ml of interferon (<i>Tau,</i> IFNT or Universal, UIFN) for 20 h prior and 2 h after being infected by BTV-8 (dark red, square), BTV8-ΔNS4 (red, triangle), BTV-1 (blue, square) and BTV1-ΔNS4 (light blue, triangle) viruses. Cells were infected at a MOI of 0.01. Supernatants were collected at 24, 48 and 72 h after infection, and then titrated on BSR cells by limiting dilution analysis and virus titers expressed as log₁₀ (TCID₅₀/ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed three times, each time in duplicate.</p

<i>In vitro</i> growth properties of rescued WT and ΔNS4 viruses.

<p>Growth curves of BTV-8 (dark red, square), BTV8-ΔNS4 (red, triangle), BTV-1 (blue, square) and BTV1-ΔNS4 (light blue, triangle) in cell lines derived from different species. BSR (hamster), BFAE (cattle), CPT-Tert (sheep), C6/36 (mosquito) and KC (<i>Culicoides</i>) cells were infected at a MOI of 0.05 and supernatants collected at 8, 24, 48, 72 and 96 h after infection. Supernatants were then titrated on BSR cells by limiting dilution analysis and the virus titers expressed as log₁₀ (TCID₅₀/ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. Experiments were performed independently twice, each time in duplicate, using two different virus stocks.</p