Electron micrographs of unstained, vitrified samples of HvV190S virions and VLPs.

<p>Representative cryo-micrographs of four separate samples are shown: (<b>A</b>) HvV190S virions; (<b>B</b>) VLP_C+; (<b>C</b>) VLP_C; and (<b>D</b>) HvV190S virions mixed with HK97 prohead II. Three particles in the virion sample (A, black arrowheads) appear empty and presumably have lost their genomes. In (D), the HK97 prohead II (white arrowhead) is clearly distinct from the smaller HvV190S virions (black arrowheads). The two largest particles in the field of view are HK97 particles that have spontaneously expanded (double arrowhead). The contrast in all panels has been adjusted to improve visibility of each sample.</p

Cryo-reconstructions of HvV190S particles.

<p>(<b>A</b>) Radial, color-coded, surface view along a twofold axis of the HvV190S virion reconstruction. A pair of similar features (outlined in white) correspond to raised portions of the two capsid subunits in one asymmetric unit of the “T = 2” capsid. (<b>B</b>) Same as (A), with the front half of the density map eliminated to show the particle interior (left) and with the genome density computationally removed to show the inner surface of the capsid (right). An innermost, fifth shell of RNA on the left side of the panel is not visible because its intensity level is close to that of noise in the density map, and the threshold used to render the map was set to a value slightly higher than this; however, a radial density plot of the virion density map (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225.s003" target="_blank">Figure S3</a>) suggests the presence of this fifth shell of RNA density. White arrowheads point to two different views of the channel located at each five-fold axis. (<b>C</b>) Planar, equatorial density projection (one pixel or ∼1.07 Å thick) of the HvV190S virion cryo-reconstruction, with features of highest and lowest density depicted in black and white, respectively. Representative two-, three-, and fivefold axes of symmetry that lie in the equatorial plane are indicated. The black arrowhead points to the channel at one fivefold axis and the dashed red oval encircles the density features that span the space near the I2 axis between the capsid and genome. (<b>D</b>) Same as (A), for the VLP_C+ (left half) and VLP_C (right half). (<b>E</b>) Inner surfaces, as in (B), for the VLP_C+ on the left and the VLP_C on the right. (<b>F</b>) Same as (C), for the VLP_C+ (left) and VLP_C (right). The scale bar in (A) is the same for all panels and the color bar specifies radii in Å units.</p

3D image reconstruction statistics for HvV190S.

1<p>Number of micrographs selected for image processing.</p>2<p>Number of particle images included in each 3D reconstruction.</p>3<p>Range of objective lens underfocus settings.</p>4<p>Estimate of resolution achieved in 3D reconstruction based on FSC_0.5 and FSC_0.143 criteria <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225-vanHeel1" target="_blank">[56]</a>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225.s002" target="_blank">Figure S2</a>.</p

Radial density projections of the HvV190S virion.

<p>(<b>A</b>) Enlarged view of upper left quadrant shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat-1003225-g003" target="_blank">Figure 3C</a> with arcs drawn at radii corresponding to the density projections shown in panels B (high radius) and I (low radius). (<b>B–I</b>) Spherical, one-pixel (∼1.07 Å) thick sections from the HvV190S virion cryo-reconstruction are shown at progressively lower radii as indicated in each panel, with highest and lowest density features depicted in black and white, respectively. Panel F corresponds to the radius (r = 183 Å) where the inter-subunit interactions are most prevalent and where the capsid-region densities are centered (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225.s003" target="_blank">Figure S3</a>).</p

Western Blot analysis of the capsid proteins and RdRp of the HvV190S virion and VLPs.

<p>Purified HvV190S virions and VLP_c+ and VLP_c were subjected to immunoblot analysis using a CP-specific antiserum (left panel) or an RdRp-specific antiserum (right panel).</p

Segmentation of the HvV190S virion capsid.

<p>(<b>A</b>) View along a twofold axis of the segmented capsid with the A- and B-subunits colored in blue and orange, respectively. Each of the subunits in one asymmetric dimer is outlined with a quadrilateral polygon. (<b>B</b>) Enlarged view of one asymmetric dimer. (<b>C</b>) Superposition of segmented A- and B-subunits shown in four different orientations, beginning with a view as seen from outside the particle (identical to the A subunit shown in panel B) and ending with a view from inside the capsid (far right). Predominant differences occur at the proximal (black arrowheads) and distal (red arrowheads) tips of the two subunits and in a pair of parallel helices (dashed white oval) that follow a curved trajectory in the A-subunit but a straighter, laterally displaced trajectory in the B-subunit.</p

Secondary structure of the ScV-L-A capsid protein and predicted structures of the CPs for HvV190S and eleven other viruses.

<p>For ScV-L-A, the secondary structure for the N-terminal 651 aa of the CP (cyan) is derived from the crystal structure (PDB ID 1M1C, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225-Naitow1" target="_blank">[30]</a>) and is predicted for the C-terminal 29 aa (red). The α-helix, β-strand, and random coil segments are represented schematically as rectangles, arrows, and lines, respectively, for ScV-L-A and all predicted structures. The six strands that comprise the prominent β-sheet in ScV-L-A (see text) are highlighted in grey. All secondary-structure predictions were made with the PSIPRED server (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#s4" target="_blank">Materials and Methods</a>). The predicted HvV190S secondary structure is rendered in black and six other, representative members of the <i>Victorivirus</i> genus are colored purple. Representative members of the <i>Giardiavirus</i>, (GLV), <i>Leishmaniavirus</i> (LRV-1), and <i>Trichomonasvirus</i> (TvV-1) genera are colored green and two unclassified viruses (EbRV1 and IMNV) are colored orange. The stretch of generally two or three adjacent helices predicted in each virus CP that may form part of a conserved, structural core is highlighted in yellow. The N-termini of all CP diagrams are left justified, and the helices (H) and strands (S) in ScV-L-A and HvV190S are labeled sequentially. Note that H19 and S31 of ScV-L-A are predicted elements whereas all the others (H1–18 and S1–30) were identified in the crystal structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003225#ppat.1003225-Naitow1" target="_blank">[30]</a>.</p