Transferrin receptor binds virus capsid with dynamic motion

Abstract

Canine parvovirus (CPV) is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Cross-species transmission of CPV occurs as a result of mutations on the viral capsid surface that alter the species-specific binding to the host receptor, transferrin receptor type-1 (TfR). The interaction between CPV and TfR has been extensively studied, and previous analyses have suggested that the CPV-TfR complex is asymmetric. To enhance the understanding of the underlying molecular mechanisms, we determined the CPV-TfR interaction using cryo-electron microscopy to solve the icosahedral (3.0-Å resolution) and asymmetric (5.0-Å resolution) complex structures. Structural analyses revealed conformational variations of the TfR molecules relative to the binding site, which translated into dynamic molecular interactions between CPV and TfR. The precise footprint of the receptor on the virus capsid was identified, along with the identity of the amino acid residues in the virus-receptor interface. Our "rock-and-roll" model provides an explanation for previous findings and gives insights into species jumping and the variation in host ranges associated with new pandemics in dogs.

Keywords: CPV; TfR; cryo-EM structure; dynamic; host jump.

Fig. 1.

Cryo-EM analysis of CPV in complex with TfR. (A) Representative image of the CPV–TfR complex micrographs. (A, Insets) The receptor density associated with the capsid structure was identified. (Scale bar, 50 nm.) (B) Surface-rendered map (gray) of the CPV capsid was reconstructed at 3.0-Å resolution using 60-fold symmetry averaging. The icosahedral symmetry axes were marked. (C) Superimposition of the VP2 structures for the capsid in the CPV–TfR complex (cyan) and the noncomplexed CPV capsid (PDB ID code 2CAS) (orange). VP2s are depicted as ribbon diagrams.

Fig. 2.

SMR identifying the asymmetric structure of the CPV–TfR:Tf complex. (A) Surface rendering of the virus–receptor complex structure. The capsid was radially colored from the center according to the color key, whereas the receptor molecule was colored light blue. (B) Fitting of the TfR (blue and orange) and 2 Tfs (gold and green) into the cryo-EM density showed that the tip of the apical domain of TfR contacts the capsid surface. B, Upper and Lower represent 90° rotated views around the y axis.

Fig. 3.

Focused 3D classification with symmetry-expanded orientation data characterizing the receptor binding sites based on the degree of receptor occupancy. (A) A simulation of 60 TfR:Tf heteromers bound to the CPV capsid was calculated by applying icosahedral symmetry operators to the bound TfR molecule in the symmetry-mismatch map (Fig. 2). The targeted volume occupied by the receptor was colored in blue; the 2 adjacent heteromers around the 5-fold axis were colored in orange; those across the symmetry axis were colored in green; those around the 3-fold symmetry axis were colored in yellow; and all other heteromers were colored in tan. (B) The transparent spherical mask applied for the target volume with occupying receptor colored in blue. (C) Five 3D-class reconstructions are shown in gray and surface rendered. The first class had full receptor densities. Classes 2 and 3 had partial densities corresponding to an unoccupied target volume plus the nearby orange receptors in A and B. The other classes had only some overlapping densities as shown. (D) A histogram for number of heteromers bound on each capsid. The average number of receptors bound to each capsid was 3.7.

Fig. 4.

Asymmetric 3D reconstruction of the CPV–TfR:Tf complex using multiple orientations. (A) Surface-rendered 3D reconstruction using all orientations selected in Fig. 3C for each virus particle. The surface was colored according to the key as in Fig. 2. (B) Local resolutions of the receptor:Tf heteromer and adjacent capsid surface. Two orientation views of the receptor density (180° rotated) showed attenuation of the local density. The surface was colored according to the local resolution color key.

Fig. 5.

Focused 3D classification of the TfR:Tf cryo-EM density. (A) The 7 3D classes, where nos. 1–5 have 2 Tfs bound to the TfR, while in classes 6 and 7, only 1 Tf is bound. The numbers of each class collected from the complete dataset are indicated. (B) Superimposition of the 5 classes (nos. 1–5) are shown. B, back-side view; F, front view; S, right-side view.

Fig. 6.

The localized reconstruction of the receptor density and fitted model identifying the TfR footprint on the CPV surface. (A) A 6.7-Å resolution cryo-EM density map for the bbj-TfR was generated by localized reconstruction image processing without symmetry. The intermediate resolution map was used to refine the bbj-TfR atomic model without the Tf bound. The TfR 3D map and model (with monomers colored in blue and red ribbons) were superimposed (A, Left) and rotated by 90° (A, Right). Three glycosylation sites with extra densities were shown by atoms in yellow spheres and labeled (A, Left). The local 3D map was surface rendered and colored transparent gray. The Tf density was removed to allow visualization. (B) The refined TfR model fitted into the asymmetric complex map. The sharpened map for CPV capsid (gray) and the fitted TfR model (blue and red) was surface rendered.

Fig. 7.

The fitting of the TfR structure identifies the dynamic interactions between CPV and TfR. (A and B) The refined bbj-TfR structure was fitted into the 5 cryo-EM maps. Fitted structures for class 3 are depicted. The 2 CPV VP2 chains and TfR are shown as ribbon diagrams and colored in blue, green, and orange, respectively. Side chains of the consistent contacts as well as Asn-93 and Ala-300 of CPV residues and TfR residues 216, 218, 207, 222, 304, and 384 are shown as stick models and labeled. (C) The roadmap of the CPV capsid surface with TfR footprint indicated. All of the receptor-contacting residues are highlighted by thick black lines. The consistent and variable contacts are further highlighted in red and yellow lines, respectively. CPV residue 93 is marked by white lines. The capsid was radially colored according to the color map, and 1 asymmetric unit is indicated by a triangle with icosahedral symmetry axis marks. (D) The capsid-contacting residues on the model of the bbj-TfR molecule. The TfR was surface rendered, and each monomer was colored differently (top). The residues that are seen to make consistent and variable contacts with the capsid were colored red and yellow, respectively. The large red surface area includes residues from the βII-1/βII-2 loop in addition to Val-213 and Leu-222.