The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication

Abstract

Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 A resolution. The capsid protein has a high content of rod-like densities characteristic of alpha-helices, forming a repeated alpha-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the alpha-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.

FIG. 1.

T=1 capsid protein structures, based on X-ray and cryo-EM data. T=1 capsids of Penicillium chrysogenum virus (PcV) (this study), L-A virus, rotavirus, bluetongue virus (BTV), rice dwarf virus (RDV), orthoreovirus, cytoplasmic polyhedrosis virus (CPV), Penicillium stoloniferum virus F (PsV-F), picobirnavirus (PBV), and φ6 phage are viewed along a 2-fold axis of icosahedral symmetry (center row). PcV CP half-protein A (this study), Gag (PDB accession no. 1m1c; 680 residues), VP2 (PDB accession no. 3kz4; 880 residues), VP3 (PDB accession no. 2btv; 901 residues), P3 (PDB accession no. 1uf2; 1,019 residues), λ1 (PDB accession no. 1ej6; 1,275 residues), VP1 (EMDB accession no. 3cnf; 1,333 residues), PsV-F CP (PDB accession no. 3es5; 420 residues), PBV CP (PDB accession no. 2vf1; 590 residues), and P1 (PDB accession no. emd-1206; 769 residues) are shown from a top view (top row). Side views of the same structures are shown (bottom row; the T=1 shell exteriors are to the right). α-Helices are represented by blue cylinders and β-sheets by yellow planks for cryo-EM-based structures. SSE of the φ6 CP were determined as described in Materials and Methods, based on the published 7.5-Å-resolution nucleocapsid cryo-EM structure (PDB accession no. emd-1206).

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).

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.

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.

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.

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.

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.