Structural basis for engagement of Western Equine Encephalitis Virus with the PCDH10 receptor

Fig. 1. Structure of WEEV VLPs alone and in complex with PCDH10-EC1-EC2. a The colored surface representations of cryo-EM maps of WEEV VLPs alone (left panel) and in complex with PCDH10-EC1-EC2 (right panel). The densities of WEEV E1 and E2 proteins are colored pale cyan and light blue, whereas the densities of PCDH10-EC1 are colored red. The 5-fold, 3-fold, and 2-fold icosahedral axes are indicated by a pentagon, triangles, and an oval, respectively. The i3 spike and q3 spike are indicated by the labels “i3” and “q3” respectively. b The equatorial cross-sections of WEEV VLPs alone (left panel) and in complex with PCDH10-EC1-EC2 (right panel) in radial distance color scheme. The radial distances are indicated by the bar. The density of PCDH10-EC1 is separately colored red for highlighting in the right panel. c, dCryo-EM maps achieved by the final refinement focusing on the q3 spike in WEEV VLPs alone (c) or in complex with PCDH10-EC1-EC2 (d). The densities are shown as a colored surface in two perpendicular views respectively.

Fig. 2. Interaction of WEEV VLP with PCDH10-EC1. a–c Structures of two WEEV E2-E1 heterodimers in complex with a PCDH10-EC1. aOne E2-E1 heterodimer is shown as a colored cartoon, while the adjacent heterodimer (E1’-E2’) is covered by a molecular surface. The color scheme for E1/E2 domains and PCDH10-EC1 is as following: E1_I, white; E1_II, light gray; E1 fusion loop, orange; E1_III, gray; E1_ecto and E1_TM, deep gray; E2_A, pale cyan; E2_β, light teal; E2_B, deep teal; E2_C, sky blue; E2_TM, deep blue; PCDH10-EC1, red. b The E1/E2 residues that interact with PCDH10-EC1 are colored in red on the molecular surface with labels in an enlarged view. Two E2-E1 heterodimers are both covered by molecular surfaces. The boundary of two E2-E1 heterodimers is roughly indicated by a dotted line. c PCDH10-EC1 is covered by a molecular surface with its interacting residues for contact with E1, E2, and E2’ in white, pale cyan, and light teal, respectively. The interaction regions 2-4 are indicated by dashed frames. d–g Details for the virus-receptor interaction. The interacting regions 1 (d), 2 (e), 3 (f), and 4 (g) are presented in enlarged views. All polypeptides are shown as cartoon diagrams.…

Fig. 3. Binding affinities of PCDH10-EC1-EC2 or its mutants with WEEV VLPs. a The binding and disassociation curves of WT PCDH10-EC1-EC2 and a representative mutant PCDH10-Q1A, -D22A, -N40A, -R42A, -H76A, -E82A, -L85A, and -Q89R with WEEV 71V1658 VLPs and WT PCDH10-EC1-EC2 with WEEV 71V1658 VLPs with 0, 6.25, 12.5, 25.0 mM EGTA are shown. The calculated KD values are displayed in each corresponding panel. The displayed results were obtained from a single technical replicate and are representative of two biologically independent experiments. The equilibrium dissociation constant (KD) was calculated as the mean (n = 2). b Summary of the measured binding affinities. The KD value for each sample is the mean value of two independent measurements. kon, association rate constant; kdis, dissociation rate constant; ND, too weak interaction cannot be detected.

Fig. 4. Cell-based assays for the impacts of PCDH10 mutants on WEEV infection. HEK293T cells stably expressing indicated PCDH10 proteins were infected with SINV-WEEV (71V1658) (MOI = 0.5) for 9 h. Infection was monitored by fluorescence microscopy (a) or flow cytometry (b). Expression of the indicated proteins was confirmed by immunoblots (c). Scale bars, 50 µm (a). Data are mean ± SD from 3 biological repeats, and p-values are from an unpaired two-tailed Student’s t test (b). At least three independent experiments were performed with similar results, and representative images are shown (a, c). Source data are available as a Source Data file.

Fig. 5. Strain-specific residues analysis and comparative analysis of receptor binding of encephalitic alphaviruses. a Comparative analysis of position 153 of the E2 protein. Left panel, sequence alignment of position 152/153 of E2 proteins encoded by WEEV strains as labeled in the figure. Group A, B1, B2, and B3 are indicated by light pink, light blue, yellow, and light purple backgrounds. Red words denote the strictly conserved residues, whereas black words denote the non-conserved residues. Right panel, the interaction of E2L153 with PCDHL85/L87. Residues are shown as colored sticks covered by the cryo-EM densities as gray meshes. b Comparative analysis of position 89 of PCDH10. Left panel, sequence alignment of position 89 of PCDH10 from Homo sapiens, Mus musculus, Equus caballus, and Passer montanus. Red words denote the strictly conserved residues, whereas black words denote the non-conserved residues. Right panel, residue PCDHQ89 and its environment WEEV residues E2P28 and E2P148’/V149’. Residues are shown as colored sticks. c BLI analyses of the binding of human PCDH10-EC1-EC2 to WEEV Imperial 181 VLPs and its mutant E2Q153L. d BLI analyses of the binding of avian PCDH10-EC1-EC2…