Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses

Abstract

The structure of recombinant virus-like particles of malabaricus grouper nervous necrosis virus (MGNNV), a fish nodavirus isolated from the grouper Epinephelus malabaricus, was determined by electron cryomicroscopy (cryoEM) and three-dimensional reconstruction at 23-A resolution. The cryoEM structure, sequence comparison, and protein fold recognition analysis indicate that the coat protein of MGNNV has two domains resembling those of tomato bushy stunt virus and Norwalk virus, rather than the expected single-domain coat protein of insect nodaviruses. The analysis implies that residues 83 to 216 fold as a beta-sandwich which forms the inner shell of the T=3 capsid and residues 217 to 308 form the trimeric surface protrusions observed in the cryoEM map. The structural similarities between fish nodaviruses and members of the tombusvirus and calicivirus groups provide significant new data for understanding the evolution of the nodavirus family.

FIG. 1.

CryoEM micrographs of frozen-hydrated samples of MGNNV (a) and PaV (b) recorded at an underfocus of ∼2.7- and ∼2.9-μm, respectively. Note the surface feature of particles in panel a.

FIG. 2.

Fourier shell correlation between two reconstructions, each independently computed by using half of the images. The correlation starts to fall below 0.5 at a spatial frequency of ∼0.043, which corresponds to a resolution of 23 Å.

FIG. 3.

CryoEM maps of MGNNV at 23-Å resolution (a) and PaV at 22 Å resolution (c). (b and d) Cutaway views of panels a and c, respectively. Panels a and b are colored radially. The densities for the protrusions (∼154 to 192 Å), the inner shell of the capsid (∼112 to 154 Å), and the RNA (<112 Å) are gold, green, and blue, respectively. In panel d, the RNA density of PaV is dark purple. In panel a, an icosahedral asymmetric unit is outlined by a triangle, and the positions of the icosahedral five-, three-, and two-fold axes are indicated by numbers. The quasi-two-fold axes are centered between the five- and three-fold axes. Bar, 100 Å.

FIG.4.

Radial density distribution of the cryoEM maps of MGNNV (bold line) and PaV (thin line) derived by spherically averaging the density in the maps. The layers of density corresponding to the protrusions, the inner shell of the capsid, and the RNA in MGNNV map are indicated by I, II, and III, respectively.

FIG. 5.

(a) Cross-sectional views of the particle envelopes of MGNNV (left) and PaV (right) defined by the cryoEM density. The refined TBSV model is superimposed with the MGNNV density and the X-ray atomic model of PaV with the PaV density. In both panels, two icosahedral asymmetric units are shown. Subunits A, B, and C are colored blue, red, and green, respectively, and the RNA duplex in the PaV model is colored yellow. (b) Comparison of one subunit of TBSV (left) that was fitted into the MGNNV density in panel a with the equivalent PaV subunit (right). The lack of connectivity between the two domains of TBSV reflects the lack of obvious density connecting the domains in the cryoEM map of MGNNV, suggesting that the connection is made by a single, extended polypeptide. (c) A view down the two-fold axis of the MGNNV density with the TBSV model superimposed. The view emphasizes the strong trimer association of the protruding domains around the quasi-three-fold axes. The C termini of subunits are indicated by stars, and the positions of icosahedral three- and five-fold axes are indicated by 3 and 5, respectively. (d) Cross section of the TBSV model superimposed on the cryoEM density of the full particle of MGNNV viewed down the five-fold axis.

FIG. 6.

Alignment of MGNNV and TBSV coat protein sequences by 3D-PSSM. Although there is no significant sequence identity between the coat proteins of the two viruses, the pattern of secondary structures of MGNNV is rich in β-elements and matches that of TBSV to a reasonable extent. The secondary structures of MGNNV (red) are predicted by 3D-PSSM, and those of TBSV (blue and light blue) are assigned based on the crystal structure. The α-helices and β-strands are shown as rods and arrows, respectively. In TBSV, the β-strands forming the N-terminal β-sandwich domain are colored light blue. The green triangle near residue 216 indicates the boundary between the putative two domains of MGNNV.