The T=1 Capsid Protein of Penicillium chrysogenum Virus Is Formed by a Repeated Helix-Rich Core Indicative of Gene Duplication

Virion preparation.PcV virions were purified from Penicillium chrysogenum strain ATCC 9480 by two cycles of differential centrifugation and a rate zonal centrifugation in a sucrose density gradient, as described elsewhere (11).

Cryo-EM.For cryoelectron microscopy (Cryo-EM), samples (5 μl) were applied to one side of a holey carbon grid, blotted, and plunged into liquid ethane, as described previously (43, 59). Full and empty PcV particles were imaged together under low-dose conditions (∼10 e⁻/Ų) in a Tecnai G2 electron microscope operating at 200 kV and equipped with a field emission gun, using a nominal magnification of ×50,000.

Image processing.General image-processing operations were performed using Bsoft (http://lsbr.niams.nih.gov/bsoft/ [25]), Xmipp (http://xmipp.cnb.csic.es/ [60]), and Spider (http://www.wadsworth.org/spider_doc/spider/docs/ [63]), as described previously (12, 43). Graphic representations were produced by using the Chimera visualization software package (http://www.cgl.ucsf.edu/chimera/ [50]).

A Zeiss TD scanner was used to digitize micrographs at a 7-μm step size to yield a 1.4-Å pixel size in the specimen. A total of 56 micrographs with a 0.9- to 3.3-μm defocus range, determined using Bshow (http://lsbr.niams.nih.gov/bsoft/bshow/bshow.html [25]) were selected for processing. X3d (15) was used to extract and normalize 6,322 and 4,530 individual particle images for PcV empty and full particles, respectively. Initial estimates of origin and orientation parameters for each image were determined using polar Fourier transform (pft) procedures (6) with the previously published PcV capsid structure (11), low pass filtered to 30 Å, as a starting model. These parameters were improved using consecutive cycles of PFT refinement of origin and orientation and subsequent computation of a 3D reconstruction and assessment of resolution. At the end of each refinement cycle, two independent reconstructions were computed using the Em3dr2 Fourier-Bessel program (http://people.chem.byu.edu/belnap/pft3dr/pft2_em3dr2.html [10]), and the new resolution limit was assessed. The subsequent refinement cycle included all Fourier data to this resolution limit. Refinement progressed until no further improvement in resolution was achieved. At the end of refinement, 5,692 and 4,156 particle images were included in the empty and full PcV capsid reconstructions, respectively.

Amplitude decay was calculated using the spatial frequency components from the cryo-EM maps (with an effective resolution determined by the Fourier shell correlation [FSC] criterion) and the X-ray L-A capsid map (PDB accession number 1m1c). The decay profile of the cryo-EM maps was then adjusted to match the profile of the X-ray maps, and the fitted function was applied to the cryo-EM maps in the frequency range from 245 Å to the maximum resolution achieved, and a soft low-pass filter was applied. In addition, amplitude decay was calculated and corrected with the Embfactor program (19), with similar results. These sharpening methods, used for identification and interpretation of high-resolution structural features, are analogous to adjusting the temperature factor (or B factor) of the two structures. Resolution was assessed by FSC between independent half-data-set maps, applying a correlation limit of 0.5.

Structural analysis.The structural asymmetric unit boundaries were established by contouring the map at different σ levels, based on its compactness and contacts with neighboring densities. To segment the asymmetric subunit and the half subunits, a dummy atom model was manually generated in Chimera and iteratively refined to avoid overlapping between subunits or loose density. To test that the selected density corresponded to a single asymmetric unit, the complete capsid map was restored in each cycle after applying icosahedral symmetry to the selected density. Using a similar approach, we deduced that the asymmetric unit is formed by two elongated ellipsoid-like structures.

The secondary structure features in the PcV capsid subunit were identified and modeled using the AIRS programs (http://ncmi.bcm.tmc.edu/software/AIRS [5]), which provides a graphic interface to the SSE Hunter and SSE Builder in the Chimera visualization software package. Secondary structural elements (SSE) were displayed as virtual reality modeling language (VRML) cylinders and planar faces (α-helices and β-sheets, respectively), and prototypical ribbon-style helices were constructed as well.

PcV CP elements A and B were initially aligned manually between both VRML SSE models in Chimera, using the ∼37-Å-long α-helices as a reference. These results were verified twice by computational structure comparison analysis between prototypical helix models generated for each subunit using LGA (local-global alignment) (http://predictioncenter.org/local/lga/lga.html [70]) with a root mean square (RMS) deviation of 1.53 Å, a level of structural similarity (LGA_S) of 14.33%, and an alignment quality score (LGA_Q) of 2.15 and by exhaustive six-dimensional search between the segmented map halves using the Foldhunter program (30) (correlation coefficient [CC] = 0.43). These approaches consistently yielded essentially the same results.

The L-A capsid protein Gag (20) X-ray structure was manually aligned to the matched VRML SSE models of PcV CP elements A and B, treated as a rigid body. The PcV 37-Å long α-helices were used as an initial anchor position for the L-A virus monomer, whose orientation was manually refined to optimize the number of overlapping SSE between both structures. The spatial arrangement for both capsid proteins in the shell was maintained. These alignments were verified using LGA, with RMS deviations of 1.67 and 1.72 Å for half-proteins A and B, respectively.

Segmentation and analysis of dsRNA densities were carried out graphically with Chimera. A dsRNA model was built using a dsRNA 34-bp fragment from BTV (PDB accession number 1h1k) (17), adjusted manually and optimized in Chimera.

Secondary-structure predictions.Secondary structure predictions were generated with the following programs (web addresses are indicated): UniProt, http://www.uniprot.org/; PsiPred, http://bioinf.cs.ucl.ac.uk/psipred/; Jnet, http://www.compbio.dundee.ac.uk/www-jpred/; Porter, http://distill.ucd.ie/porter; Sable, http://sable.cchmc.org; Gor, http://gor.bb.iastate.edu; Yaspin, http://www.ibi.vu.nl/programs/yaspinwww/; Profsec, http://www.predictprotein.org/; and GeneSilico, http://genesilico.pl/meta2/.

Protein structure accession numbers.3D reconstructions are deposited in EMBL-EBI (accession numbers emd-1610 and emd-1611 [full and empty particles, respectively]).

PcV capsid three-dimensional structure at 8-Å resolution.PcV particles were purified and analyzed by cryo-EM as described previously (11). Full and empty particles were imaged together (Fig. 2A), and their 3D reconstructions were calculated separately. The final resolution of the reconstruction for full PcV virions was estimated to be 8.0 Å based on a Fourier-shell correlation (FSC) threshold of 0.5 (and 8.9-Å resolution for empty particles) (Fig. 2B). The central sections of the 3D maps showed no marked structural differences between full and empty capsids in the protein shell (Fig. 2C and D). This interpretation was further supported when full and empty particles were subjected to joint refinement cycles; the estimated resolution of the combined 3D map was 7.5 Å, indicating that both maps are virtually identical (not shown). The quality of the information provided by the resolution profile is better for the full-capsid 3D reconstruction; the map images shown are taken from this reconstruction.

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FIG. 2.

Three-dimensional cryo-EM of PcV virions. (A) Representative cryoelectron micrograph of native PcV virions (2-μm underfocus; first zero of the contrast transfer function at 22 Å). Bar = 50 nm. (B) Assessment of the resolution of full and empty PcV reconstructions. FSC resolution curves were calculated for the full (blue) and empty (red) capsids. Each set of particle images was subdivided randomly into two subsets, and independent reconstructions were computed from these data. The resolutions at which the correlations dropped below 0.5 and 0.3 are indicated. For the 0.5 threshold, the values for full and empty PcV were 8.0 and 8.9 Å, respectively; values for the 0.3 threshold were 7.15 and 8.0 Å, respectively. 1/A, 1/Å. (C, D) Central sections from the 3D reconstructions of full (C) and empty (D) capsids, viewed along a 2-fold axis. Protein and/or RNA are dark. The two protein shells are virtually identical, and the RNA density of the full capsid is quite low compared to that of the protein shell. (E) Stereo view of the radially color-coded outer surface of the full capsid, viewed along a 2-fold axis of icosahedral symmetry. The most prominent features are 12 outward-protruding pentamers. The map is contoured at 2 σ above the mean density. Bar = 100 Å. (F) Surface view of PcV virion, viewed along an icosahedral 5-fold axis and showing the five CP structural subunits in a pentamer (green, orange, blue, pink, and yellow).

The particle molecular architecture is essentially as previously described at lower resolution (11). The capsid's diameter is 400 Å, and the protein shell is 48 Å thick. The most prominent features of the T=1 capsid were 12 outwardly protruding pentons that correspond to the pentameric positions of the icosahedral lattice (Fig. 2E). The enantiomorph maps shown in this study were chosen based on preliminary X-ray analysis that, in addition, matched well with our docking analysis (below). The structural asymmetric unit boundaries were established by contouring the map at different levels, based on its compactness and contacts with neighboring densities (Fig. 2F). To confirm that the selected density corresponded to a single asymmetric unit, the complete capsid map was fully restored after applying icosahedral symmetry to the selected density.

At 8 Å resolution, numerous rod- and sheet-like densities were found on the relatively uneven outer surface, similar to L-A virus. This is in contrast with the smooth outer surface of the reovirus particles, in which the CP has a plate-like structure.

Structural duplication in the PcV capsid protein.The asymmetric unit is formed by two ellipsoid-like structures with roughly similar morphology, although they differ somewhat in size (Fig. 3 A, blue and yellow). The asymmetric unit is a rhomboidal prism, ∼46 Å high and ∼130 Å long (Fig. 3B). The smaller part makes up 43% of the total unit volume (blue; half-protein A) and the larger one represents 57% (yellow; half-protein B). These elements are arranged in two sets of five; five half-protein A structures directly surround the icosahedral 5-fold axis, and five half-protein B structures are intercalated between them, forming a pseudodecamer (Fig. 3A). This quaternary organization is similar to the 120-subunit T=1 shell in reoviruses and totiviruses (Fig. 1).

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FIG. 3.

Structure of the PcV T=1 capsid and model of the capsid protein fold. (A) Surface-shaded virion capsid viewed along an icosahedral 2-fold axis with capsid protein (CP) elements in cyan and yellow; boundaries for two CPs are outlined in red. Icosahedral symmetry axes are numbered. (B) Segmented asymmetric unit (PcV CP monomer). The dashed line highlights the rhomboidal shape. Protein halves A (cyan) and B (yellow) are indicated. Red symbols indicate icosahedral symmetry axes. (C) PcV CP secondary structural elements (SSE), using the same color scheme and orientations as in panel B. Cylinders, α-helices; planks, β-sheets. The red arrow indicates translation direction to superimpose half-protein B on A. Black arrows indicate the ∼37-Å-long α-helices of both PcV CP elements.

The SSE Hunter program showed a high α-helical content for the PcV CP; we identified 16 α-helices and 5 β-sheets in the half-protein A structure and 24 α-helices and 7 β-sheets in the larger half-protein B structure (Fig. 3C). We used various sequence-based SSE prediction methods (PsiPred [32], Jnet [14], Porter [51], Sable [2], Gor [62], Yaspin [39], and Profsec [58]) on the PcV CP sequence (982 amino acids) to test the correlation with our structural subunit model (Fig. 4). SSE predictions from the PcV CP sequence indicate a high α-helical content, as directly visualized in our cryo-EM map and identified with the SSE Hunter program. In addition to a consensus SSE prediction compatible with our fold model, we found an unstructured central region that divides the PcV CP into two parts, reflecting a structurally disordered region (amino acids ∼550 to ∼650). The SSE gap in the middle of the protein sequence was confirmed by the consensus prediction of protein order with the GeneSilico metaserver (35). The unstructured region might represent the linker sequence between the two unequal halves of the PcV CP.

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FIG. 4.

Secondary structure consensus prediction for the PcV CP. The PcV CP sequence was obtained from the UniProt database (982 amino acids; accession no. Q8JVC1). Several SSE prediction methods (PsiPred, Jnet, Porter, Sable, Gor, Yaspin, and Profsec) were used to correlate our model of the structural subunit. A consensus SSE prediction was obtained by simple majority at each sequence position. The results were very similar to the consensus SSE prediction obtained with the GeneSilico fold prediction metaserver. The secondary structure gap in the middle of the protein sequence (segment Glu555-Ser650) reflects a structurally disordered region, as confirmed by the consensus prediction of protein order with the GeneSilico metaserver, and divides the PcV coat protein into two parts. Arrow, β-chain; bar, α-helix.

Both PcV CP elements appeared to have a similar ∼37-Å-long α-helix tangential to the capsid surface (Fig. 3C, black arrowheads). Careful inspection showed that both protein halves, though not identical, have substantial resemblance. When one of the halves (treated as a rigid body) is superimposed on the other by an ∼45-Å translation (Fig. 3C; the red arrow indicates translation direction), the relative spatial locations of 13 α-helices and two planar regions are close and require only minor local adjustment (Fig. 5A; also see Movie S1 in the supplemental material). Structurally preserved SSE between both PcV CP halves consist of two domains, domain I with a characteristic bundle of four α-helices, located toward the 5-fold axis, and domain II, which contains the longest α-helix, as well as other SSE. Whereas the conserved core establishes lateral contacts with the neighboring units, the interface between halves A and B is less similar, with a larger number of variations. This analysis indicates that despite a lack of sequence similarity, the two PcV CP elements have a comparable structural signature, suggesting ancestral gene duplication.

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FIG. 5.

Structural matching of the PcV CP elements. Comparison of PcV and L-A capsid proteins. (A) Superimposed views of conserved SSE in PcV protein halves A (cyan) and B (yellow); the relative spatial locations of 13 α-helices and two planar regions are very close. PcV protein elements are subdivided into domains I and II. (B) L-A virus Gag structure. Shown are the polypeptide chain path (top view), as a gradient from blue (N terminus) to red (C terminus) (left); a cylinder-and-plank representation of Gag, as for PcV CP, with 17 α-helices and 10 β-sheets (center); and selected Gag SSE (9 α-helices and 2 β-sheets) (right). (C) Superimposed views of helical and planar regions of selected Gag SSE (red) and PcV half-protein A (blue). This structural matching preserves the spatial orientation of both units.

Structural comparison of PcV and L-A capsid proteins.With the exception of the ∼430-amino-acid CP of the partitivirus PsV-F, the PcV CP structural halves are the smallest polypeptides of the dsRNA virus T=1 proteins (Gag is almost twice as large and VP3, P3, CPS, and λ1 are almost four times as large as the PcV CP C-terminal element). They might represent a fold that evolved from the T=1 CP ancestral fold to the highly varied structures observed today. If this hypothesis is correct, “structural remnants” of the conserved PcV motif might be found in the modern T=1 CP.

PcV CP was compared to L-A virus Gag CP for several structural and functional reasons: (i) PcV and LA viruses both infect lower eukaryotes and lack an extracellular phase in their life cycles; (ii) they share not only an uneven outer surface, in contrast to the smooth outer surface of reovirus particles, but also a roughly similar side view (Fig. 1); and (iii) PcV and L-A CP have no outer shell, whereas Reoviridae T=1 cores serve as a template to prime the assembly of the T=13 surrounding capsid.

Overlaying L-A Gag (Fig. 5B) on either of the PcV elements, while maintaining the same spatial arrangement in the shell, showed many SSE with similar spatial distributions, including nine α-helices (of a total of 13) and two β-sheets (Fig. 5C; also see Movie S2 in the supplemental material). These structural similarities are located in the Gag N terminus (residues 1 to 414); the entire C-terminal region (residues 415 to 680) would be a terminal insertion to this conserved fold. This structural matching, in which CP orientations are maintained (i.e., the outer and inner surfaces are coincident), indicates a shared motif for these two fungal virus T=1 CPs.

dsRNA packaged within the capsid.We studied the organization of the packaged RNA at relatively low resolution using data from our 3D cryo-EM studies. Analysis of PcV full capsids showed that the inner surface of the protein shell is in close contact with the underlying density shell that is ascribed to encapsidated dsRNA (Fig. 6A). Spherically averaged radial density plots computed from the 3D reconstructions of full and empty PcV particles indicated that, as a result of these extensive interactions, there is almost no space between the two layers (the average distance of the empty space is 9 Å). The innermost RNA shells are more diffuse, as the RNA density is quite low compared to that of the protein shell (Fig. 6B). There is clear correspondence in the peaks centered at a radius of ∼180 Å, suggesting little if any variance in the overall architecture of the protein shell.

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FIG. 6.

Genomic dsRNA within the PcV virion particle. (A) A PcV 50-Å-thick slab. Capsid shell coloring is the same as in Fig. 3, contoured at a lower contour threshold (1.0 σ above the mean density) to highlight the locations of the dsRNA densities. dsRNA (green) is seen as approximately four concentric layers contoured at 1.0 σ. There are numerous contacts between the inner surface of the capsid and the outer surface of the nearest dsRNA layer. (B) Radial density profiles from 3D maps of full and empty PcV particles. Both profiles are practically superimposable at the protein shell (radius ∼162 to 200 Å). A difference map (full subtracted from empty capsid) was calculated by arithmetic subtraction of the density values. (C) A transverse section, 1.4 Å thick, taken from the 3D map of a full PcV capsid, parallel to but displaced ∼7 Å from the central section viewed along a 2-fold axis (darker, denser). (D) Same section as in panel C, but contoured at 1.0 σ above the mean (protein in yellow and RNA in green), which shows the six types of CP-dsRNA interactions (1 to 6). The two adjacent boxes show magnified views of the interactions between the inner surface of the capsid and the dsRNA outermost layer. (E) PcV capsid asymmetric unit (shown as wire frames, contoured at 2.8 σ, viewed from the inside) with the SSE as shown in Fig. 2C (cyan and yellow for half-proteins A and B, respectively). dsRNA interacts with six defined areas of the capsid inner surface (dashed ovals, 1 to 6). Observe that the cross-section size of the contacting areas is larger than that corresponding to an α-helix. Black symbols indicate icosahedral symmetry axes.

The existence of RNA-protein contacts was reported previously (11); here we define the sites (SSE) on the icosahedral protein shell involved in these interactions. The interactions of each CP half with the RNA show overall similarities (for example, three interacting areas along each one). This is consistent with the suggested structural duplication, although there are differences that reflect structural changes in the protein halves. There exist at least 360 interacting areas in total, as found after imposing icosahedral symmetry (Fig. 6C and D). These observations agree with the overall basic character of PcV CP (theoretical pI, 8.24). Specific RNA-protein interactions are evident in six defined areas of the PcV monomer inner surface (Fig. 6E, labeled 1 to 6) that will be ascribed to specific sequences when high-resolution data are available. These areas are located within the conserved SSE, whereas the interface between the CP elements (with many variations) is free of interactions.

Due to the numerous icosahedral positions that interact with the genome, the outermost RNA layer has strong features of icosahedral symmetry conforming to an icosahedral cage (Fig. 7A). A difference map calculated by subtracting the full from the empty capsid shows this layer as a conspicuous peak beneath the shell protein peak. The bulk of the genome density appears diffuse toward the center of the core, in two or three concentric layers; this density weakening suggests greater RNA disorder at greater distances from the capsid shell (Fig. 6B). RNA-protein contacts appear to form tracks on the inside of the CP shell, resembling those described in the X-ray structure of BTV (see Fig. 4 in reference 22). This layer appears to be made of tube-like densities with dimensions of dsRNA helices. A-form helices of dsRNA fitted remarkably well into the cryo-EM density map (Fig. 7B). Whereas tubular densities are concentric around the 5-fold axis in BTV, in PcV they converge into the 5-fold axes and connect with neighboring strands from adjacent 5-fold axes, describing a T=1 genome shell (Fig. 7A). These dsRNA strands reinforce the connectivity between two monomers that contributes to adjacent pentamers (Fig. 7C) and, therefore, increase capsid stability.

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FIG. 7.

Outer layer of dsRNA. (A) Genome density ordered into an icosahedral cage in close contact with the capsid inner surface. The capsid protein has been stripped away, showing the top half of the first layer of ordered dsRNA density. (B) A-form helices of dsRNA docked into the tubular densities corresponding to the dsRNA density. (C) Close-up view down a 2-fold axis from inside, showing two adjacent structural subunits colored as in Fig. 3. The phosphate backbone is traced as a red ribbon for the two dsRNA A-form strands. Icosahedral symmetry axes are indicated.