Structure of avian orthoreovirus virion by electron cryomicroscopy and image reconstruction - PubMed (original) (raw)

Structure of avian orthoreovirus virion by electron cryomicroscopy and image reconstruction

Xing Zhang et al. Virology. 2005.

Abstract

Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the "non-fusogenic" mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these "fusogenic" orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 A, showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric lambda-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric sigma-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks "hub-and-spokes" complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T=13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric mu-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features.

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Figures

Fig. 1

CryoEM and 3DR of ARV virions. (A) Raw cryoelectron micrograph of well-separated ARV-138 virions. The arrowhead denotes a probable region of degradation in the outer capsid. Scale bar, 100 nm. (B) Plot of the Fourier shell correlation (FSC) as a function of resolution for the averaged electron density map of ARV-138 virions. Based on a conservative threshold criterion (solid horizontal line for FSC = 0.5), the effective resolution of the averaged map is near 14.6 Å. Based on a less stringent noise-limited criterion (dashed horizontal line for FSC = 0.143; Rosenthal and Henderson, 2003), the effective resolution is near 12.1 Å.

Fig. 2

Surface views and central cross-sections of the 3DRs of ARV and MRV virions. (A, B) Radially color-coded surface views of ARV-138 (A) and MRV-T3D (B). The same radial color map was applied to each particle, as indicated by the legend at right; the numbers indicate radial distances from the center of each particle, in Å. The positions of one P2 channel and one P3 channel are labeled in each particle. (C, D) Central cross-sections of ARV-138 (C) and MRV-T3D (D). Darker shades indicate greater density in the averaged map. Positions of icosahedral twofold (2f), threefold (3f) and fivefold (5f) symmetry are marked by black lines and labeled. Specific densities attributable to each of the seven structural proteins are indicated by white arrows and labeled with the protein names in each particle. Concentric rings of density, attributable to the dsRNA genome segments in the particle interiors, are indicated by curving dashed lines. Scale bar in panel D applies to all panels, 20 nm.

Fig. 3

Radial sections of the 3DRs of ARV and MRV virions. The row of numbers across the middle of the figure indicates the radii, in Å, at which the sectional views above and below each number were generated. Fivefold-symmetrical densities attributable to pentamers of the λC protein in ARV-138 (A, C, E) or the λ2 protein in MRV-T3D (B, D, F) are surrounded by a black pentagon in each panel. Threefold-symmetrical densities attributable to trimers of the µB protein in ARV-138 or the µ1 protein in MRV-T3D are labeled with white letters in panels A to D; the different letters (Q, R, S, and T) denote the four symmetrically distinct positions that the µB or µl trimers occupy within the T = 13 outer capsid. A region centered on one P3 channel of each particle, and shown in magnified view in Fig. 4 is surrounded by a white circle in panels C and D; arrowheads denote densities in the channel that are absent in ARV-138, but present in MRV-T3D. Ovoid densities attributable to monomers of the σA protein in ARV-138 or the σ2 protein in MRV-T3D are labeled with white letters in panels E and F; the different letters (2f, 3f, and 5f) denote the three symmetrically distinct positions that the σA or σ2 monomers occupy between the λ1 (inner) and µl (outer) layers, at or surrounding the icosahedral twofold (2f), threefold, (3f), or fivefold (5f) axes. Scale bar in panel F applies to all panels, 20 nm.

Fig. 4

Magnified views of one P3 channel each from the 3DRs of ARV and MRV virions. The region of each particle shown here is the same as that surrounded by a white circle in Figs. 3C and D P3 channel densities absent in ARV-138 (A) are present and have a “hub-and-spokes” appearance in MRV-T3D (B). Scale bar in panel B applies to both panels, 1 nm. (C) An alignment of the C-terminal sequences of the homologous µl, µB, and VP4 outer-capsid proteins from various isolates of MRV (T1L, type 1 Lang; T2J, type 2 Jones), ARV, and aquareovirus (AqRV) (GCR, grass carp reovirus; GSR, golden shiner reovirus), respectively (Noad et al., in press). Residues identically conserved in at least two of the three groups are bolded. The stop codon that terminates each protein is shown as an asterisk. A GenBank accession number for each sequence is listed at the right.

Fig. 5

Stereo views showing fits of the X-ray crystal structures of MRV proteins λ2 and µ1into the 3DR of the ARV virion and its enantiomer. The density map of the ARV-138 virion (gray surface) was rendered with laevo (A, C) or dextro (B) handedness in its T = 13 outer capsid. (A, B) The view is centered on the icosahedral fivefold axis. The crystal structure of the λ2 pentamer (Reinisch et al., 2000) is shown in ribbon format, with each of the five subunits in a different color (green, red, cyan, blue, yellow). Attempts to reposition the λ2 pentamer within the density map of the dextro enantiomer led, in all instances, to significantly poorer fits than that shown for the laevo enantiomer. (C) The view is centered on a P2 channel adjacent to the icosahedral fivefold axis near the bottom right corner. The crystal structure of the µ1trimer (Liemann et al., 2002) is shown in ribbon format, with each of the three subunits in a different color (purple, white, orange), and is fit into each of the four µ1trimer positions surrounding the P2 channel. The crystal structure of one adjacent λ2 subunit is also shown in ribbon format (green).

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Structure of avian orthoreovirus virion by electron cryomicroscopy and image reconstruction - PubMed (original) (raw)