As anticipated from the difference in appearance of the fish and insect nodaviruses in high-contrast, unprocessed images of frozen hydrated particles (Fig. ), the cryoEM structure of MGNNV differed remarkably from those of insect nodaviruses, such as Pariacoto virus (PaV) (Fig. ). Like that of the insect nodaviruses, the surface morphology of MGNNV is consistent with a T=3 quasi-equivalent lattice; however, the details of the morphology and the radial density distribution of MGNNV differ dramatically from those of PaV (Fig. and ). The maximum diameter of MGNNV is approximately 380 Å, significantly larger than the PaV capsid at 360 Å. The density distribution of the MGNNV map shows two shells, at radii corresponding to protein, separated by low density. The outer shell is between radii of 154 and 192 Å with a maximum at 168 Å; the inner shell is between radii of 112 and 154 Å with a maximum at 135 Å. The MGNNV map is contoured at a level such that the resultant volume of these two shells is equal to the expected volume of its protein capsid, which is formed by 180 copies of a 38-kDa subunit. It is noteworthy that the interior boundary of the inner shell (112 Å) of MGNNV and PaV are nearly coincident, and this defines the interior limit of the β-sandwich domain of PaV. Density with a radius of less than 112 Å (Fig. and ) is probably predominately RNA.
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), (more ...)
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 (more ...)
The outer shell of MGNNV is composed mainly of the large protrusions located at the quasi-three-fold axes. These protrusions are much more prominent than those of PaV (Fig. ). The inner shell is relatively uniform, and the protrusions are separated from it by nearly a 12-Å void, indicating that the connection between the domains is probably a single, extended polypeptide chain that is not visible in the cryoEM map.
The crystal structures of PaV and Flock house virus showed that the protrusions at highest radius are formed by three two-stranded β-sheets related to each other by quasi-three-fold symmetry (5
) (Fig. ). These β-sheets are formed by insertions between strands of the canonical viral β-sandwich that forms the contiguous protein shells of the insect nodaviruses, and the strands are twisted together about the quasi-three-fold axes to form the surface protrusions. The inner and outer regions of the insect nodaviruses, however, display continuous density in contrast to the density gap between the outer and inner shells of protein in MGNNV.
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 (more ...)
Superposition of the cryoEM map of MGNNV with the atomic model of PaV derived from the crystal structure showed relatively good agreement for the region occupied by the contiguous β-sandwich shell in PaV. However, there was poor agreement in the outer radial region (Fig. ). The protrusions at the quasi-three-fold axes in MGNNV have much larger volume than could be accounted for by the three twisted β-sheets at the surface of the PaV structure (Fig. ). Thus, these protrusions must contain more protein that may form individual domains. Moreover, the PaV model could not account for differences in density observed in the MGNNV reconstruction at the icosahedral and quasi-two-fold symmetry axes. Significant density exists at the icosahedral two-fold axes between protrusions. However, there is only weak density at the corresponding regions between protrusions related by the quasi-two-fold symmetry (Fig. ). This density difference implies different patterns of contacts between protrusions at icosahedral and quasi-two-fold axes.
Both the cryoEM reconstruction and the crystal structure of PaV revealed that an ordered portion of the viral RNA forms a dodecahedral cage composed of 30 segments of RNA duplex closely associated with the capsid (20
). In the reconstruction of MGNNV, there is no significant density adjacent to the capsid that can be interpreted as duplex RNA. However, the density at lower radius (<112 Å) may be attributed to the cellular RNA randomly packaged inside the capsid and/or portions of the coat protein, e.g., the N-terminal basic-residue-rich segments which are likely to interact with RNA (Fig. ).