Properties of reconstructions of virion, procapsid, and mutant particles of PRD1.

a<p>Determined at full width half the maximum of the peak.</p>b<p>Based on radially averaging the central section of each reconstruction and comparing the averaged intensity of the membrane layer to the capsid layer.</p><p>Properties of reconstructions of virion, procapsid, and mutant particles of PRD1.</p

The unique vertex organization.

<p>(A) The side view, (B) the central slice view, and (C) the top slice view of the segmented unique vertex in the mature virion. The cut-through view (C) (location labeled as the orange dashed line in (A) and (B)) shows the 12 arms (numbered 1 to 12) of the central part of the unique vertex and the extra surrounding densities. (D) The side view, (E) the side slice view, and (F) the top slice view of the segmented transmembrane density in the procapsid. The top slice view (F) (location labeled as the orange dashed line in (D)) shows the 12 arms of the structure organized as hexameric dimers (numbered 1 to 6 in circles). The density of the unique vertex in the procapsid (grey) is shown on that of the mature virion (cyan) as a side slice view (G) and top slice view (H).</p

Delineating the boundary between the unique vertex and the surrounding major capsid proteins.

<p>(A) The ribbon representation of the regular 5-fold vertex structure (PDB code 1W8X). The penton P31 is shown in dark blue and the surrounding peripentonal P3 trimers are shown in five colors, each of which belongs to one asymmetric unit. (B) The top view and (C) the side slice view of the segmented unique vertex density (grey) surrounded by ten P3 trimers. The unique vertex replaces the penton and five peripentonal P3 trimers (shown as transparent gray density in (A)). (D) The electrostatic potential surface of the surrounding peripentonal MCP P3s was calculated with APBS and colored in Chimera ranging from red (negative) to blue (positive) with central slice view.</p

Reconstructions of two packaging vertex deficient particles at 22 and 18 Å resolutions without icosahedral symmetry imposition.

<p>(A) A typical micrograph of Sus526 mutant particles (P6<i>⁻</i>, P9<i>⁻</i>, P20<i>⁻</i> (P22<i>⁻</i>), DNA-P8<i>⁻</i>). (B) The top view and (C) the central slice view of the Sus526 reconstruction. (D) A typical micrographs of Sus42 mutant particles (P6<i>⁻</i>, P9<i>⁻</i>, (P20<i>⁻</i>), P22<i>⁻</i>, DNA-P8<i>⁻</i>). (E) The top view and (F) the central slice view of the Sus42 reconstruction.</p

Reconstruction of PRD1 virion at 12 Å resolution without icosahedral symmetry imposition.

<p>(A) A typical micrograph of the PRD1 virions. (B) The top view and (C) the central slice view of the reconstruction. (D) The unique vertex occupies one of the 12 pentonal positions and interacts with the capsid proteins at its outer edge. (E) The other 11 vertices have a regular 5-fold structure. (F) The crystal structure of the MCP P3 of PRD1 (PDB code 1W8X, chain B) fitted in the corresponding segmented density in the cryo-EM map, allowing the boundary delineation of the unique vertex complex and the MCPs surrounding it.</p

Wt and mutant PRD1 viruses and their properties.

a<p><i>S. enterica</i> serovar Typhimurium LT2 suppressor strain harboring plasmid pLM2 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002024#pbio.1002024-Mindich3" target="_blank">[74]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002024#pbio.1002024-Winston1" target="_blank">[75]</a>.</p>b<p><i>S. enterica</i> serovar Typhimurium LT2 DS88 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002024#pbio.1002024-Bamford7" target="_blank">[73]</a>.</p>c<p>Presence of the protein in the particle is rather uncertain based on biochemical analysis.</p><p>Wt and mutant PRD1 viruses and their properties.</p

Reconstructions of the procapsid and Sus621 particle at 14 and 19 Å resolutions without icosahedral symmetry imposition.

<p>(A) Typical micrograph of the procapsid virus particles (P9<i>⁻</i>, DNA-P8<i>⁻</i>) containing P20, P22, and P6 without P9 and DNA-P8 complex at the unique vertex. (B) The top view and (C) the central slice view of the procapsid reconstruction. (D) Typical micrograph of the Sus621 virus particles (P6<i>⁻</i>, P9^50%, DNA-P8<i>⁻</i>) containing P20, P22, and 50% P9 without P6 and DNA-P8 complex at the unique vertex. (E) The top view and (F) the central slice view of the Sus621 reconstruction. The unique vertex (grey) in either map only shows transmembrane densities but no ordered densities at the capsid region.</p

Inhibition of HSV-1 replication by <i>UL29</i>-specific siRNA molecules.

<p>HaCaT and U373MG cells on 96-well plates were transfected with 10 pmol of indicated siRNA molecules or water. After 4 h the cells were infected with 1000 pfu of HSV-1 and incubated for 44 h. (<i>A</i>) Dilutions of the HaCaT and U373MG supernatant, collected 48 h after transfection (44 h post infection) were assayed for released virus by plaque formation on Vero cell culture. (<i>B</i>) The relative expression of the target HSV-1 gene <i>UL29</i> was measured by qRT-PCR from samples of the infected cultures. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. are shown for six replicates. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 and (¤) p<0.05 against the controls; (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other siRNA transfections. NT, no transfection; mock, transfection with water; eGFPGD, anti-eGFP siRNA pool; eGFPS, single-site anti-eGFP siRNA; UL29GD, GD-digested anti-UL29 siRNA pool; UL29HD, HD-digested anti-UL29 siRNA pool; UL29L, 27-nt single-site anti-UL29 siRNA; UL29S, 21-nt single-site anti-UL29 siRNA.</p

Innate immunity responses to siRNA pools and single-site siRNA molecules.

<p>HaCaT and U373MG cells were transfected with either 1 (light grey) or 10 (dark grey) pmol of the indicated siRNA molecules; 1 pmol of 88-bp dsRNA (black bar) or water (control, grey bar); or left untreated (control, white bar). The expression levels of <i>IFN-β</i> (<i>A</i>), <i>IFN-λ1</i> (<i>B</i>) or <i>ISG54</i> (<i>C</i>) were assessed by qRT-PCR 8 h, 24 h or 48 h post transfection. Values were normalized to the <i>GAPDH</i> housekeeping gene and shown on a logarithmic scale. The mean values+S.D. of at least two independent experiments, each with a minimum of three biological replicates, are shown. Data were compared by Mann-Whitney U-test. The statistical significance is indicated as (×) p < 0.01 or (¤) p < 0.05 against the controls; (**) p < 0.01 or (*) p < 0.05 against a group of comparison; (#) p < 0.01 against all other transfections. UL29GD, GD-digested anti-<i>UL29</i> siRNA pool; UL29HD, HD-digested anti-<i>UL29</i> siRNA pool; UL29L, 27-nt chemically synthesized anti-<i>UL29</i> siRNA; UL29S, 21-nt chemically synthesized single anti-<i>UL29</i> siRNA. No significant signal was detected for IFN-λ1 mRNA in U373MG cell culture (B, right panel) if no dsRNA was applied (non- and mock-transfected cells).</p

Proposed model for PRD1 procapsid assembly, genome packaging and virus maturation.

<p>(A) Viral membrane-associated proteins are incorporated into the host cytoplasmic membrane, including integral membrane proteins P20 and P22. (B) P20 and P22 form the transmembrane pore in the newly-assembled viral membrane. (C) Various capsid-associated proteins then assemble along the viral membrane to form the viral capsid, while P6/P9 assembles onto the transmembrane pore (P20/P22) and forms the unique packaging complex, which completes the assembly of the procapsid. (D) The newly synthesized viral genomic DNA is then packaged through the unique vertex fueled by the hydrolysis of ATP. (E) Along the genome packaging, the membrane is pushed against the icosahedral protein shell as the last step in the virus maturation.</p