Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1

Abstract

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.

Importance: Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

FIG 1

Biochemical and cryo-EM analyses of RnQV1 strains W1075 and W1118. (A) Coomassie blue-stained SDS-11% PAGE gels of purified full and empty RnQV1-W1075 (left) and empty RnQV1-W1118 (right) virions used for cryo-EM data acquisition. P4 and P4-related bands are indicated in gray and P2 and P2-related bands in black. Molecular mass markers (10⁻³ Da) are on the left. (B) Cryo-EM of full RnQV1 strain W1075 (the arrows indicate two empty capsids). (C) Cryo-EM of empty RnQV1 strain W1118. Bar = 50 nm.

FIG 2

Three-dimensional cryo-EM of RnQV1 virions. (A) Assessment of the resolution of full (W1075) and empty (W1118) RnQV1 reconstructions. FSC resolution curves were calculated for full (blue) and empty (red) capsids. Each set of particle images was subdivided randomly into two subsets, and independent reconstructions were computed from the data. Resolutions for which correlations were <0.3 are indicated. For the 0.3 threshold, the values for full and empty RnQV1 capsids were 8.2 and 9.1 Å, respectively. (B and C) Central sections from the 3D reconstruction of full (B) and empty (C) capsids, viewed along a 2-fold axis. Protein and RNA are dark. The two protein shells are virtually identical, and the RNA density of the full capsid is seen as concentric circles inside the capsid. (D) 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 120 outward-protruding densities (orange). The map is contoured at 2.5 σ above the mean density. Bar = 100 Å. (E) Surface-shaded virion capsid viewed along an icosahedral 5-fold axis showing the five A (blue) and B (yellow) structural subunits in a pentamer. (F) Inner surface of the RnQV1-W1075 capsid (for clarity, only the density between 145- and 210-Å radii is shown). Icosahedral-symmetry axes are indicated (red symbols).

FIG 3

Structure of the RnQV1 capsid and model of the heterodimer fold. (A) Segmented asymmetric unit (A-B heterodimer). The dashed line highlights the rectangular shape. Subunits A (blue) and B (yellow) are indicated. The map is contoured at 2.5 σ above the mean density. Icosahedral-symmetry axes are indicated (red). The insets highlight P and S domains of CP (height is indicated). (B) SSE of subunits A and B, using the color scheme and orientations in panel A; cylinders, α-helices; planks, β-sheets. The arrows indicate the subunit A α-helix that forms the 5-fold axis (middle) and the subunit B β-sheet that forms the 3-fold axis (right). (C) RnQV1 capsid pores at the 5-fold axis (∼16-Å diameter) and the 3-fold axis (∼18-Å side).

FIG 4

Sequence alignment and secondary-structure consensus prediction for RnQV1 P2 and P4 CP amino acid sequences. The sequences of P2 (1,356 amino acids for W1075; 1,357 amino acids for W1118) (A) and P4 (1,061 amino acids for W1075; 1,059 amino acids for W1118) (B) were obtained from the UniProt database [H1ACC6, M1VMJ0, H1ACC8, and M1VHN2, respectively). Several SSE prediction methods (PsiPred, Jnet, Porter, Sable, Gor, Yaspin, and Profsec) were used to test correlation with our models of the structural subunits. A consensus SSE prediction was obtained by simple majority at each sequence position. Identical residues (white on red background) and partially conserved residues (red) are indicated. The arrows indicate β-strands, and the spirals indicate α-helices.

FIG 5

Genomic dsRNA within the RnQV1 virion particle. (A) A 50-Å-thick RnQV1-W1075 slab. Capsid shell coloring is the same as in Fig. 2, contoured at 1.2 σ; dsRNA (green) is represented as three concentric layers contoured at 1.0 σ. (B) Radial density profiles from 3D maps of full (W1075) and empty (W1118) RnQV1 particles. Both profiles are superimposable at the protein shell (radius, ∼162 to 235 Å). A difference map was calculated by arithmetic subtraction of the density values for both structures (full minus empty capsid; dashed line). Small differences in the protein shell are indicated at radii 175 to 195 and 205 to 220 (black arrows); major differences in the genome region are seen as density peaks at radii of 80, 113, and 147 Å (green arrows). The radial density profile from the 3D map of full ScV-L-A virions is also shown (L-A).

FIG 6

Analytical ultracentrifugation analysis of RnQV1-W1075. Sedimentation velocity experimental data (A) and sedimentation coefficient distribution obtained by c(s) analysis (B) of empty capsids (solid line) and virions or full particles (dashed line).

FIG 7

Comparison of T=1-based inner cores of dsRNA viruses. (Top) Asymmetric units (top views) of RnQV1 (A), ScV-L-A (B), and PcV (C). RnQV1 A and B subunits are shown in blue and yellow, respectively, to indicate that they are distinct proteins; ScV-L-A A and B subunits are shown in blue, since they are conformers of the same protein; and PcV A and B “subunits” are shown in blue (with the boundaries of A outlined in red), as they are two similar covalently bound domains of a single CP. (Bottom) Surface-shaded capsids of RnQV1, ScV-L-A, and PcV viewed along an icosahedral 2-fold axis. To indicate that they are similarly organized capsids, the A subunits (closer to the 5-fold axis) are shown in blue and the B subunits (intercalated between A subunits) in yellow.