Capsid Structure of dsRNA Fungal Viruses

Abstract

Most fungal, double-stranded (ds) RNA viruses lack an extracellular life cycle stage and are transmitted by cytoplasmic interchange. dsRNA mycovirus capsids are based on a 120-subunit T = 1 capsid, with a dimer as the asymmetric unit. These capsids, which remain structurally undisturbed throughout the viral cycle, nevertheless, are dynamic particles involved in the organization of the viral genome and the viral polymerase necessary for RNA synthesis. The atomic structure of the T = 1 capsids of four mycoviruses was resolved: the L-A virus of Saccharomyces cerevisiae (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1). These capsids show structural variations of the same framework, with 60 asymmetric or symmetric homodimers for ScV-L-A and PsV-F, respectively, monomers with a duplicated similar domain for PcV, and heterodimers of two different proteins for RnQV1. Mycovirus capsid proteins (CP) share a conserved α-helical domain, although the latter may carry different peptides inserted at preferential hotspots. Insertions in the CP outer surface are likely associated with enzymatic activities. Within the capsid, fungal dsRNA viruses show a low degree of genome compaction compared to reoviruses, and contain one to two copies of the RNA-polymerase complex per virion.

Keywords: PcV; PsV-F; RnQV1; ScV-L-A; capsid protein; capsid structure; dsRNA virus; mycovirus; viral lineage; virus evolution.

Figure 1

T = 1 capsid protein X-ray- and cryo-EM-based structures. Top row: T = 1 capsids of bluetongue virus (BTV), orthoreovirus, rice dwarf virus (RDV), cytoplasmic polyhedrosis virus (CPV), rotavirus, grass carp reovirus (GCRV), picobirnavirus (PBV), ϕ6 phage and ϕ8 phage, L-A virus of Saccharomyces cerevisiae (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1), viewed along a two-fold axis of icosahedral symmetry (center row). BTV VP3 [2] (PDB accession number 2btv; 901 residues), λ1 [41] (1ej6; 1275 residues), P3 [42] (1uf2; 1019 residues), VP1 [19] (3cnf; 1333 residues), VP2 [43] (3kz4; 880 residues), GCRV VP3 [22] (3k1q; 1027 residues), PBV CP [23] (2vf1; 590 residues), ϕ6 P1 [44] (4btq; 769 residues) and ϕ8 P1 [45] (4btp; 792 residues), Gag [26] (1m1c; 680 residues), PcV CP domain A [46] (3j3i; 498 residues; complete CP 982 residues), PsV-F CP [29] (3es5; 420 residues) and RnQV1-W1118 P2 and P4 [36] (5nd1; 972 and 1005 residues, respectively), shown from top view. Bottom row: side views of the same structures (T = 1 shell exterior at right).

Figure 2

ScV-L-A T = 1 capsid protein; X-ray-based structure. (A) T = 1 capsid of ScV-L-A viewed along a two-fold axis of icosahedral symmetry, showing the Gag subunits A (blue) and B (yellow), with the boundaries of the asymmetric unit outlined in red. Numbers indicate icosahedral symmetry axes. (B) Atomic model of a Gag dimer (1m1c; 680 residues). Icosahedral symmetry five- (pentagon), three- (triangle), and two-fold (oval) axes are indicated in black. (C) T = 1 CP dimers of BTV (2btv) and ϕ6 (4btq). The structural unit is an asymmetric dimer in which subunits A and B are oriented in parallel with numerous side contacts. Symbols indicate icosahedral symmetry axes. (D) Side view of a Gag monomer (T = 1 shell exterior top). His154, the active site for decapping activity is indicated.

Figure 3

PcV T = 1 capsid protein; cryo-EM-based structure. (A) T = 1 capsid of PcV viewed along a two-fold axis of icosahedral symmetry, showing the N-terminal domain A (1–498, blue), the linker segment (499–515, red), and the C-terminal domain B (516–982, yellow), with the boundaries of an asymmetric unit outlined in red. Numbers indicate icosahedral symmetry axes. (B) Top view of the atomic of the PcV CP (3j3i; 982 residues). Symbols indicate icosahedral symmetry axes. (C) PcV capsid protein is a structural duplication. Superimposed A and B domains (white segments indicate non-superimposed regions for both domains). (D) Sequence alignment of domains A (blue) and B (yellow) resulting from Dali structural alignment. α-helices (rectangles) and β-strands (arrows) are rainbow-colored from blue (N terminus) to red (C terminus) for each domain. Triangles represent non-aligned segments (sizes indicated): the orange triangle indicates the single “hotspot” on the outer capsid surface. Strictly conserved residues are on a red background and partially conserved residues are in a red rectangle.

Figure 4

PsV-F T = 1 capsid protein X-ray-based structure. (A) T = 1 capsid of PsV-F viewed along a two-fold axis of icosahedral symmetry, showing the CP subunits A (blue) and B (yellow), with the boundaries of an asymmetric unit (a quasi-symmetric A-B dimer) outlined in red. Numbers indicate icosahedral symmetry axes. (B) Top view of the atomic model of a PsV-F CP dimer (3es5; 420 residues). Symbols indicate icosahedral symmetry axes; red oval indicates a local two-fold symmetry axis. (C) Side view of a PsV-F CP dimer. The arch (green) and shell domains (red) are indicated. For clarity, the second subunit is shown in grey. (D) T = 1 CP dimers of PBV (2vf1), BMV, brome mosaic virus (1js9), and cowpea chlorotic mottle virus (CCMV; 1cwp). Dimers are related by a local quasi-two-fold symmetry axis (red oval), and show molecular swapping. BMV and CCMV are plant ssRNA viruses with T = 3 capsids, but their CP (with a β-barrel domain) can assemble into 120-subunit capsids that show a quaternary organization similar to that of PsV-F and PBV.

Figure 5

RnQV1 T = 1 capsid cryo-EM-based structure. (A) T = 1 capsid of RnQV1 viewed along a two-fold axis of icosahedral symmetry, showing P2 (blue) and P4 (yellow). Boundaries for an asymmetric unit are outlined in red. Numbers indicate icosahedral symmetry axes. (B) Top and (C) side views of the atomic models of P2 (blue; 972 residues) and P4 (yellow; 1005 residues) (5nd1). The last visible P2 C-terminal residue is located on a P4 surface crevice. (D) Sequence alignment of P2 (blue) and P4 (yellow) resulting from Dali structural alignment. α-helices (rectangles) and β-strands (arrows) are rainbow-colored from blue (N terminus) to red (C terminus) for each protein. Dashed rectangles indicate favorable insertion sites, triangles represent non-aligned segments (sizes indicated).

Figure 6

Structural homology of mycovirus T = 1 CP. The PcV CP A domain (PcV-A, left, center) was structurally aligned with ScV-L-A Gag (right, center), and P2 (top, center) and P4 (bottom, center) with PcV-A and ScV-L-A Gag. Center rainbow-colored structures indicate conserved secondary structure elements within the dsRNA viruses. PcV-A is aligned with ScV-L-A Gag (blue and pink, center). P2 is aligned with PcV-A (blue and light blue, top left) and with ScV-L-A Gag (blue and pink, top right). P4 is aligned with PcV-A (yellow and blue, bottom left) and P4 with ScV-L-AL-A Gag (yellow and pink). Total numbers of secondary structural elements with close relative spatial locations are indicated.

Figure 7

dsRNA virus T = 1 capsid inner surfaces with electrostatic potentials. (A) PcV and (B) RnQV1 capsid inner surfaces viewed along a two-fold axis of icosahedral symmetry. The inner surface charge representations of these capsids show the distribution of negative (red) and positive (blue) charges. Note the numerous electropositive areas in PcV. Arrows indicate the capsid pores at the five-fold (black) and three-fold (blue) axes. Boxes: magnified views of the five- (top) and three-fold (bottom) pores showing charge distribution on the channel walls. (C) T = 1 capsids of ScV-L-A, PsV-Fe, rabbit picobirnavirus, and rotavirus (from left to right), viewed along a two-fold axis of icosahedral symmetry. dsRNA and packaged proteins (such as RNA polymerases) were removed computationally.