High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions

Figures

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  Fig. 1.. Immunization experiments and nsEMPEM analysis of polyclonal immune complexes. (A) Assembly of the CV capsid (left) and structure of the asymmetric unit (right). CV-A21 structure [PDB ID 1z7s (23)] was used to make the figure. VP1–4 are colored differently and labeled for clarity. (B) Schematic representation of the workflow used to generate pAb samples (top) and immunization regimen details (bottom). (C) Representative EM micrograph (top) and 2D class averages (bottom) of CV-A21 viral particles alone (left) or in complex with pAbs (as Fab fragments) isolated from CV-A21-naïve mice (middle) or mice immunized with CV-A21 (right). (D) Integrated pAb-corresponding signal in 3D maps reconstructed using the nsEM data presented in panel (C). These data are generated using Volume Tools in UCSF Chimera (24). (E) pAb-corresponding signal plotted on the surface of low-pass filtered map of CV-A21 viral capsid. Higher signal intensity corresponds to higher pAb occupancy. The icosahedral symmetry axes in panels (A) and (E) are indicated with golden-yellow pentamers (5-fold), triangles (3-fold), and ovals (2-fold).

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  Figure 2.. cryoEMPEM analysis of bound polyclonal antibodies. (A) Schematic illustration of the cryoEMPEM workflow. (B) High-resolution antigen-pAb immune complexes featuring structurally unique antibody specificities detected in cryoEMPEM dataset. Antigen is represented in gray and pAb densities are colored according to the epitope (blue and pink colors are used to represent Sites 1 and 2, respectively). The apparent global resolution of each reconstructed cryoEM map is indicated. Close-up views of the antibody–antigen interface are shown below each reconstructed map (cryoEM map—transparent gray surface; antigen—dark gray; antibodies—blue or pink). Ribbon representation is used for atomic models. The most relevant icosahedral symmetry axes are indicated with golden-yellow pentamers (5-fold), triangles (3-fold), and ovals (2-fold).

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  Fig. 3.. Antibody binding analysis based on cryoEMPEM data. (A and B) Locations of the Site-1 (A) and Site-2 (B) epitope clusters. Three VP protomers are shown for Site-1, and five protomers are shown for Site-2. For clarity, epitopes are presented only on a single protomer–protomer interface and the protomers comprising the interface are colored based on the specific VP subunit (VP1–green; VP2–yellow; VP3–salmon; VP4–dark blue). VP1’–VP4’ belong to the first protomer in the interface and VP1”–VP4” to the second protomer. Specific epitope residues are indicated below the chain ID. Ribbon representation is used in the panel. Polyclonal antibody footprints are indicated in blue (Site-1) and pink (Site-2). (C) Surface representation of the CV-A21 viral particle with Site-1 (blue), Site-2 (pink), and the receptor binding site (RBS; orange) indicated. For clarity, only a single RBS is highlighted. (D and E) Overlay of pAbC1-1 (D) and pAbC2-2 (E) antibody structures and ICAM-1 receptor structure [PDB ID: 1z7z; (23)]. (F) Surface representation of the CV-A21 viral particle colored based on sequence conservation. The location of the Site-1, Site-2, and RBS is outlined in blue, pink, and orange,…