Article Figures & Data
Figures
- FIG 1
Location of residue P73 in CNV and associated density in the 3.0-Å X-ray structure at the putative zinc binding region at the quasi-6-fold axis. The top figure is a stereo pair and represents the crystal structure of C subunit termini involved in zinc binding with the RNA interior toward the bottom of the figure. For clarity, the backbone atoms are shown in green, yellow, and mauve for the three C subunits. The model has two alternative positions for zinc based on the electron density, and these are represented by mauve spheres. At bottom is the sequence alignment of various tombusviruses in that region. CyRSV, cymbidium ringspot tombusvirus; TCV, turnip crinkle virus; MNSV, melon necrotic spot virus; RCNMV, red clover necrotic mosaic virus; CLSV, cucumber leaf spot virus; PoLV, pathos latent virus.
- FIG 2
Cryo-EM structures of the wild-type CNV in the native and swollen states and P73G in the native state. The stereo pairs are centered on the quasi-6-fold axis (Q6). The model for the native wild-type (wt) and P73G mutants is the unmodified crystal structure of CNV (5) while that of the swollen wild-type virion is the same crystal structure fitted into the density using Situs (10). The A, B, and C subunits are shown in blue, green, and red, respectively. Q3, quasi-3-fold axis.
- FIG 3
Conformational changes in CNV during the swelling process. (A) Stereo pair of the B subunit under native (green) and swollen (wheat) conditions. The models were aligned using the shell (S) domain as a guide. Notice the large movement of the P domain with respect to the S domain as the subunits are splayed apart during swelling. (B) Differences between the crystal structure at the quasi-6-fold axis and the cryo-EM structure of the wild-type swollen particle. The B and C subunits are shown in green and red, respectively.
- FIG 4
Example density of the P73G mutant cryo-EM structure under native conditions. In these stereo pairs, the A, B, and C subunits are colored blue, green, and red, respectively. Calcium ions were identified in the X-ray structure (5) and are represented by mauve spheres.
- FIG 5
Stereo pairs of electron density of the inner surface of the capsid at the quasi-6-fold axis. The top panels show the density of the crystallographic structure of CNV at the quasi-6-fold axis (5). The mauve sphere in the center represents the putative zinc ion chelated by three icosahedrally related C-chain His residues. Subsequent panels show the density of the cryo-EM structure of the wild-type (wt) CNV under native conditions (FSC1/2 of 6.8 Å) and under swollen conditions (FSC1/2 of 9.9 Å), as indicated. The model shown at the annulus is from the native, wild-type structure even though the C subunits are displaced from the quasi-6-fold axis due to the swelling. The bottom panels show the cryo-EM structure of the P73G annulus under native conditions (FSC 0.143 of 4.2 Å and FSC1/2 of 4.5 Å). While the density of the subunits around the annulus is well defined, the metal binding area at the annulus is badly disordered.
- FIG 6
Particle stability as measured by resistance to urea denaturation. The top figure shows the effects of urea on CNV mobility in a 1% agar gel made from 0.5× TB buffer, pH 8.3, that contained ethidium bromide for visualization. The loading well is located at the top of the gel. The dashed boxes denote the sections of the gel that were excised and run on an SDS-PAGE gel. The bottom figure shows an SDS-PAGE gel of the excised bands indicated in the top panel. Only the lower bands from the agar gel were analyzed by SDS-PAGE. Note that with the P73G mutant, the lower band disappears at 1 M urea and then reappears at higher urea concentrations.
- FIG 7
Negative-stain EM images of representative samples from the urea denaturation experiments. Under native conditions (i.e., pH 5.0), both the P73G and wild-type virions are uniform and monodispersed. Upon incubation in the swelling buffer, the wild-type virions expand; their cores are penetrated by stain but are still well dispersed. In contrast, the P73G particles are more heterogeneous in shape and form large aggregates. The bottom figures show the differences between the wild type and the P73G mutant under swelling conditions in the presence of 3 M urea. In the case of the P73G mutant, while the major virus band reappears in the agarose gel at 3 M urea, this sample contains only disrupted virions.
