Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release

FIG 1. Endosomal ion and 0.01% faf-BSA treatments, individually or in combination, lead to CVA9 virion expansion. (A) Real-time spectroscopy measurements of the fluorescent dye SYBR green II accessibility to the CVA9 genome in the presence of 0.01% faf-BSA in an endosomal ionic environment (green) with (dotted line) or without (solid line) RNase. Untreated CVA9 in DPBS (black) with (dotted line) or without (solid line) RNase was used as a control. (B) Real-time spectroscopy measurements of fluorescent dye SYBR green II accessibility to CVA9 genome in the presence of 0.01% faf-BSA (red) or endosomal ion composition (blue) with (dotted line) or without (solid line) RNase. The fluorescence signal under the dotted curve indicates expanded virions, whereas fluorescence signal between solid and dotted curves originated from released genome, i.e., empty capsids. (C) Effect of 0.01% faf-BSA and endosomal ions on metabolically 35S-labeled CVA9 virions as analyzed by ultracentrifugation in a 5%-to-20% sucrose gradient and subsequent fractionation. CVA9 after endosomal ionic treatment (blue) and 0.01% faf-BSA treatment (red) is shown. Nontreated CVA9 in PBS-Mg buffer was used as a control (gray).…

FIG 2. AF4 allows separation of the heterogeneous CVA9 virion population generated by 0.01% faf-BSA and endosomal ion treatments. (A) Two independent AF4 fractograms of untreated CVA9. (B) AF4 fractogram of CVA9 after 1-h treatment with 0.01% faf-BSA at 37°C. (C) AF4 fractogram of CVA9 after 3-h treatment with 0.01% faf-BSA at 37°C. (D) AF4 fractogram of CVA9 after 1-h treatment in endosomal ion environment at 37°C. (E) AF4 fractogram of CVA9 after 3-h treatment in endosomal ion environment at 37°C. (F) AF4 fractogram of CVA9 after combined treatment (0.01% faf-BSA in endosomal ionic environment) for 1 h at 37°C. In all panels, the x axis indicates time (1-mL fractions were collected at the rate of 0.5 mL/min); the y axis on the left shows light scattering (black or gray lines) and on the right the hydrodynamic radius is shown, as measured by online dynamic light scattering detector (black dots). The vertical dashed line on the left indicates the peak for intact particles and on the right the peak for expanded particles. AF4 fractions used for MS analysis are indicated by red boxes. The light scatter absorbance on all the fractograms was normalized to the highest light scatter…

FIG 3. CVA9 expansion dependency on faf-BSA and palmitate concentrations. (A) Fractionation of untreated (gray), 0.01% faf-BSA-treated (red) or 0.0005% faf-BSA-treated (orange) CVA9 after separation in a 5%-to-20% sucrose gradient using ultracentrifugation. The peak for intact virions corresponded to fractions 15 to 17, for expanded virions to fractions 12 to 14, and for empty virions to fractions 7 to 10. (B) Analysis of CVA9 treated with 0.005% faf-BSA in the presence of an excess of palmitate (pink) after ultracentrifugation in a 5%-to-20% sucrose gradient. For comparison, 0.005% faf-BSA-treated CVA9 was ultracentrifuged the same way without addition of palmitate and is shown in purple. Similar analysis of CVA9 treated with endosomal ion concentrations with (blue) or without (light blue) addition of palmitate is also shown. The peak for intact virions corresponded to fractions 15 to 18, for expanded virions to fractions 12 to 14, and for empty capsids to fractions 7 to 10. Buffer composition is shown in Table 1.

FIG 4. Workflow of focused classification applied to the faf-BSA-treated CVA9 data set to sort heterogeneity. (A) Cross-section of an initial faf-BSA-treated CVA9 reconstruction using standard 2D and 3D classifications. Features of interest at the 2-fold and 5-fold axes were enlarged, as well as the position of the hydrophobic pocket. Despite the nominal resolution at 3.5-Å resolution, the density under the 5-fold axis was much more ordered than expected. Focused classification on either the 2-fold or 5-fold axis yielded two distinct classes corresponding to intact and expanded particles, indicating that ordered density was due to contamination with intact virion particles in the reconstruction. The consensus classes were used for further refinement. (B) Cross-section of the faf-BSA-expanded CVA9 particle density map. Features at the 2-fold and 5-fold axes and the collapsed hydrophobic pocket are enlarged. (C) Cross-section of the native CVA9 particle density map obtained from a 2D class in the faf-BSA-treated CVA9 data set. In all three panels, features at the 2-fold, 5-fold, and the collapsed hydrophobic pockets are enlarged. Density maps are radially colored, in angstroms, from the…

FIG 5. Heterogeneity of CVA9 virions in different data sets after cryo-EM and single-particle analysis. (A) Analysis of the data set of untreated CVA9 resulted in intact, empty nonexpanded, and expanded virions containing the genome. (B) Data set of 0.01% faf-BSA-treated CVA9 separated into expanded empty and filled capsids, in addition to intact particles. (C) CVA9 after treatment with endosomal ionic concentrations, in addition to main class of genome-containing expanded virions, yielded a small population of intact virions and expanded empty capsids. In all panels, top view shows central plane of the refined reconstructions, and below it is the enlarged view of a surface representation of a reconstruction, shown at the 2-fold axis and clearly indicating an opening seen in expanded virions. Distribution of particle classes is indicated on the top right corner, and resolution of the reconstruction is shown below the figure. Reconstructions are colored according to radial distance, in angstroms, from the particle center (color key shown on the left) and shown at 3 σ above the mean.

FIG 6. Cryo-EM data collection and single-particle analysis. (A) Representative micrographs from untreated, 0.01% faf-BSA-treated, and endosomal ionic conditions-treated CVA9 data sets. Scale bar, 100 nm. (B) Example 2D class averages obtained in Relion 2 during processing workflow for three data sets; the 2D class average corresponding to empty virions is from the untreated CVA9 data set. (C) FSC curves of final reconstructions for intact CVA9, 0.01% faf-BSA-treated and endosomal ion-treated CVA9, and estimated resolutions at a 0.143 threshold. (D, left) Central sections of the CVA9 reconstructions, colored according to local resolution in angstroms, as shown in the color keys. Reconstructions are shown at 2 σ above mean. (Right) Central planes of the reconstructions.

FIG 7. Comparison of reconstructions and atomic models of intact, 0.01% faf-BSA-treated, and endosomal ion-treated CVA9 virions. (A) Untreated CVA9 central section through the model fitted to the reconstruction shown at 3 σ above mean. (Left) Cryo-EM density with atomic model fit; (right) cryoEM density accounted for by the atomic model (not shown) to reveal the unmodeled density. Symmetry axes are marked as follows: 5-fold, pentagon; 3-fold, triangle; 2-fold, ellipse. Enlarged view of the model at a 2-fold symmetry axis highlights the vRNA contacts with Trp38 from VP2, observed in all three reconstructions. The enlarged view on the right shows that most of the density in the capsid region was accounted for. The VP1 N terminus extends on the inside of the capsid hugging VP4, and density for a lipid factor can accommodate an atomic model of a palmitate. (B) Central section through the atomic model fitted into the reconstruction of 0.01% faf-BSA-treated CVA9. On the right, half of the density that was accounted by an atomic model was removed to better visualize the unmodeled density. Enlarged view of vRNA contacts with Trp38 from VP2 is shown. View of unmodeled density near the VP1 N…

FIG 8. Calculations of electrostatic potentials show the significant change from a positive to a negative charge on the inner surface of the capsid when the intact virion (PDB ID 8AT5), the intact virion with VP4 removed computationally prior to calculating the electrostatic potential (PDB ID 8AT5), and the expanded virion (PDB ID 8AW6) are compared down a 2-fold axis of symmetry. (A) Electrostatic potentials are shown on the outer surface of the capsid for a cluster of four asymmetric units. The proteins contributing to the electrostatic potential are shown in the schematic diagram of the virion surface and are similar for all three renderings. (B) Electrostatic potentials are shown on the inner surface of the capsid for a cluster of four asymmetric units. The proteins contributing to the electrostatic potential are shown in the schematic diagrams of the virion inner surface. Electrostatic potentials are shown according to the color key in panel A. Blue, VP1; green VP2; salmon, VP3; cyan, VP4.