Structural and functional analysis of coxsackievirus A9 integrin αvβ6 binding and uncoating

Abstract

Coxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin αv family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for αvβ6, which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating.

Fig 1

Electron micrographs and icosahedral reconstructions of CVA9 in complex with integrin. (A and B) Micrographs of CVA9 capsid alone (1.6-μm underfocus) and in complex with integrin α_vβ₆ (3.0-μm underfocus), respectively. In panels A and B, a white arrow indicates an empty particle and a black arrow indicates a filled particle. Bar, 100 nm. (C and D) Radially depth-cued isosurface representations of the filled CVA9 capsid (C) and filled CVA9 capsid-integrin α_vβ₆ complex (D) filtered to 10.3-Å resolution. (E and F) Radially depth-cued isosurface representations of the empty CVA9 capsid and empty CVA9 integrin α_vβ₆ capsid filtered to 9.9-Å resolution. In panels C to F, the view is down a 2-fold axis of symmetry. The scale bar is for the radial depth cueing. (G) Radial profiles for the empty CVA9-integrin α_vβ₆ complex (line with black dashes), filled CVA9-integrin α_vβ₆ complex (line with black dashes and dots), empty CVA9 capsid (solid line), and filled CVA9 (dotted line) (one pixel is 1.13 Å).

Fig 2

Comparison of the atomic model of CVA9 with the CVA9-integrin complex model. (A) Central cross-section view of icosahedrally symmetric filled CVA9 capsid-integrin α_vβ₆ density (left side) and central cross-section of simulated density for the CVA9 atomic model (PDB 1d4m; right side) (12, 44), with the symmetry axes marked 3f, 2f, and 5f. Protein and RNA are shown in black. Bar, 15 nm. (B) Comparison of asymmetric reconstruction of the filled CVA9 capsid-integrin α_vβ₆ complex (wire net) with the CVA9 atomic model (yellow ribbon model). The integrin density seen as a protrusion in the wire net is from a position that is symmetry related with respect to the one enforced during alignment. The integrin is on top of the VP1 C terminus shown as a red ball model of valine 284. (C) The interaction of a single protomer of CVA9 (VP1, red; VP2, green; VP3, yellow; VP4, blue) with the difference map (wire net) created by subtracting the CVA9 atomic model from the icosahedrally symmetric filled CVA9 capsid-integrin α_vβ₆ density. The density mainly represents RNA.

Fig 3

Tomographic reconstructions of the filled CVA9 capsid-integrin α_vβ₆ complex. Clear integrin density bound to virus particles can be seen in 0.76-nm-thick slices through the tomographic reconstructions. The number of integrins bound per capsid appears variable. Different conformations of integrin are seen attached to the capsids. White arrow, capsid; black arrow, integrin. Bar, 50 nm.

Fig 4

Radially depth-cued isosurface representations of the asymmetric reconstruction of the filled CVA9-integrin α_vβ₆ capsid. (A) The reference model used in an asymmetric reconstruction run in AUTO3DEM (41) viewed down a 2-fold axis of symmetry. The reference model has an integrin β-chain in the active state bound to one position adjacent to the C terminus of VP1 on the filled CVA9 capsid reconstruction. (B) The asymmetric reconstruction generated by AUTO3DEM at a 0.5 standard deviation (SD) above the mean, showing the low occupancy of the integrin in the symmetry-related positions on the capsid, where the highest signal is for the position aligned to the modeled integrin shown in panel A. The 2-fold orientation of the capsid is shown as a schematic diagram, which is then rotated to a 5-fold orientation. (C) Five-fold view of the model described for panel B with 5 equivalent positions around one 5-fold vertex marked with numbers 1 to 5. Position 1 is that enforced by the model. Position 3 has a weak signal for the integrin. (A to C) The scale bar for the radial depth cueing is shown in panel A, chosen so that the density at the radii of the integrin is shown in red.

Fig 5

Surface plasmon resonance sensograms for α_vβ₆ binding to CVA9. Data are shown for 50, 100, and 150 nM integrin injected over the CVA9 coated chip surface. The x axis is time in seconds, the y axis is the surface plasmon resonance signal in response units (RU).

Fig 6

Flexible fitting of the atomic model of CVA9 into the density map representing both the filled and empty integrin-bound capsids. (A and B) Atomic structure of CVA9 protomer (VP1, VP2, and VP3) shown fitted in a single asymmetric unit of a filled CVA9 integrin α_vβ₆ capsid density map and empty CVA9 integrin α_vβ₆ capsid density map (wire net), respectively, with the correlation value given below each map. (A and B) The density maps are shown at 2.5 standard deviations above the mean. The direction of the symmetry axes is shown. (C) Superimposed models of filled (yellow) and empty (red) fitted structures. Panel D presents a view rotated 180° with respect to panel C. Symmetry axes are marked in panels C and D. Panel E shows the G81-Y89 α-helices of neighboring VP2 moving apart in the empty fitted structure compared to the filled one, around a 2-fold axis. In panels A to E, all the atomic models are shown in ribbon form.