Molecular mechanisms of receptor recognition and antibody neutralization of coxsackievirus A6

Figures

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  Fig. 1. Development and characterization of anti-CVA6 neutralizing MAbs. a Isotype and neutralization activity of MAbs 1F4 and 3H7. Neutralization concentration was defined as the lowest antibody concentration fully preventing cytopathic effect. Neutralization IC50 values were derived from cell viability assays (b, c). b, c Neutralization potency of the MAbs against CVA6 strains 141 and HeB. Dose-response curves of the MAbs against CVA6-141 (b) and CVA6-HeB (c) were quantified via cell viability assays. An anti-SARS-CoV-2 antibody served as negative control (Ctr). Data represent mean ± SEM of four replicate wells in 96-well cell culture plates. Conc., concentration. d Binding of the MAbs to purified CVA6-HeB virions analyzed by ELISA. Data are expressed as mean ± SD from triplicate wells. e Binding kinetic analysis of the MAbs to immobilized CVA6-HeB virions by biolayer interferometry (BLI). Association and dissociation steps are divided by dotted red line. Sensorgrams show responses at serial MAb concentrations (nM range labeled). Equilibrium dissociation constants (KD) were calculated by Octet Data Analysis software. f Therapeutic efficacy of MAbs 1F4 and 3H7 against CVA6-HeB and…

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  Fig. 2. Mechanisms of action of CVA6-neutralizing MAbs. a Time of addition assay. CVA6-HeB was incubated with MAbs 1F4 or 3H7 before (Pre) or at indicated time points after (Post) viral attachment to RD cells. Viral RNA was quantified by RT-qPCR at 6 hours post-infection (hpi) and is expressed as a percentage of the virus-only control. Data are mean ± SD (n = 4 for virus-only; n = 3 for others). p = 0.0055 (1F4-Post-0.5 h); p = 0.0336 (3H7-Post-0.5 h). b Attachment inhibition assay. CVA6-HeB was incubated with serially diluted anti-CVA6 MAbs (1F4, 3H7) or a control antibody for 1 h, followed by adsorption to pre-chilled RD cells at 4 °C for 2 h. Cell-associated viral RNA was analyzed via RT-qPCR. Viral RNA levels are expressed as percentages relative to the virus-only group. and is expressed as a percentage of the virus-only control. Data are mean ± SD (n = 3). p = 0.0112 (1F4-1ng), p = 0.0030 (1F4-10ng), p = 0.0181 (1F4-100ng), p = 0.0152 (3H7-1ng), p = 0.0126 (3H7-100ng). Statistical note for a, b: Significance was determined by two-tailed Student’s t-test versus the virus-only control: ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ****, p < 0.0001. c Schematic representation of competitive…

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  Fig. 3. Cryo-EM structures of CVA6 particles before and after KRM1 binding. a, b Cryo-EM density maps of UV-inactivated CVA6-HeB mature virion (a) and empty particle (b). All maps are radially colored (scale in Å) and viewed along the two-fold axis. One icosahedral asymmetric unit is marked by a black triangle. Pentagons and triangles denote five-fold and three-fold symmetry axes, respectively. 2D classification revealed 93.0% mature virions and 7.0% empty particles. c, d Twofold axis channel structures in the CVA6 mature virion (c) and empty particle (d). VP1 (blue), VP2 (green), VP3 (red), and VP4 (yellow) are shown; this color scheme applies to all panels. Ellipses represent the two-fold axis. Conformational differences are outlined by yellow dashed rectangles. e, f Atomic models of the VP1 hydrophobic pocket in the CVA6 mature virion (e) and empty particle (f). A pocket factor (magenta stick) is fitted into the VP1 hydrophobic pocket of the CVA6 virion, with corresponding cryo-EM density (magenta mesh). g, h Central cross-sectional views (Z-axis) of CVA6 mature virion (g) and empty particle (h). i–k Cryo-EM analysis of the KRM1-treated CVA6 sample revealed a shifted composition:…

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  Fig. 4. Molecular interactions between CVA6 and KRM1. a Surface representation of a CVA6 capsid pentamer bound to KRM1, viewed along the five-fold symmetry axis. CVA6 pentamer is colored gray. KRM1 is magenta. One icosahedral asymmetric unit is marked by a triangle. Density maps displayed are unsharpened. b One KRM1 molecule interacts with two adjacent CVA6 protomers. KRM1 is shown as magenta ribbon, superimposed on cryo-EM density. CVA6 subunits are shown as ribbons and color-coded: Protomer 1 (VP1.1: light blue; VP2.1: light green; VP3.1: light red). Protomer 2 (VP1.2: blue; VP2.2: green; VP3.2: red). c Interaction of KRM1’s KR and WSC domains with adjacent CVA6 protomers. KRM1 is drawn as a cartoon with the domains color-coded: KR, cyan; WSC, orange; CUB, black. CVA6 protomers are drawn as surface. d Enlarged view of the CVA6–KRM1 interaction interface, highlighting key residues on KRM1. e–j Binding interfaces between KRM1 and specific capsid regions: e VP3.1 CD-loop, f VP3.1 C-terminus, g VP1.1 C-terminus, h VP1.2 EF-loop, i VP1.2 GH-loop, j VP2.2 EF-loop. Hydrogen bonds are indicated by gray dashed lines; salt bridges by black dashed lines; regions with both hydrogen bonds and salt…

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  Fig. 5. Cryo-EM structures of CVA6 particles in complex with 1F4 Fab. a, b Cryo-EM density maps of CVA6-HeB-1F4 in two conformations: 1F4-bound virion (a) and empty particle (b). Both maps are radially colored and viewed along the twofold axis. An icosahedral asymmetric unit is marked by a black triangle. 2D classification resolved 98.9% 1F4-bound virions and 1.1% empty particles. c, d Density maps of twofold-related protomers in the 1F4-bound virion (c) and empty particle (d), superimposed with atomic models. Conformational differences are outlined by yellow dashed rectangles. 1F4 Fab is omitted in (c) for clarity. e, f Atomic models of the VP1 hydrophobic pocket in the 1F4-bound virion (e) and empty particle (f). The pocket factor (magenta) is present in the 1F4-bound virion but absent in empty particle. Consequently, the VP1 pocket collapses in empty particle, accompanied by a significant inward shift of the VP1 GH loop (residues 223–225; red arrowhead, RMSD = 1.4 Å). 1F4 Fab is omitted in (e) for clarity. The five-fold axis is indicated. g, h Central cross-sections of the 1F4-bound virion (g) and empty particle (h) along the z-axis. Black arrows indicate 1F4 Fab density. Central…

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  Fig. 6. Cryo-EM analysis of CVA6–3H7 interactions. a, b Cryo-EM density maps of CVA6-HeB-3H7 resolved into two states: a 3H7-bound virion (98.1%) and b 3H7-associated empty particle (1.9%). c, d Structural features of the twofold axis channel in the 3H7-bound virion (c) and 3H7-associated empty particle (d). Structural differences are outlined by yellow dashed rectangles. 3H7 Fab is removed for clarity. e, f Structures of VP1 hydrophobic pocket in the 3H7-bound virion (e) and 3H7-associated empty particle (f). A pocket factor (magenta) is present in the virion but absent in the empty particle, leading to pocket collapse and an inward displacement of the VP1 GH loop (red arrowhead). 3H7 Fab is omitted in both panels for clarity. The fivefold axis is indicated. g, h Central cross-sections of the 3H7-bound virion (g) and 3H7-associated empty particle (h). Black arrows indicate 3H7 Fab density. i Atomic model of a CVA6 protomer in complex with 3H7 Fab. VH is cyan, and VL is magenta. The fivefold axis is labeled. j, k Enlarged views of interaction interfaces between CVA6 and the 3H7 VH (j) or VL (k). Gray dashed lines represent hydrogen bonds. Black dashed lines indicate salt bridges. l…

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  Fig. 7. Competitive binding of 1F4 and 3H7 to overlapping epitopes on CVA6. a Superimposed cryo-EM density maps of CVA6-1F4 and CVA6-3H7 complexes. The maps are colored by radial distance. 1F4 Fab is shown in magenta. 3H7 Fab is shown in blue. b Footprint analysis of 1F4 and 3H7 on CVA6 surface. Contour lines mark KRM1 in orange, 1F4 Fab in yellow, and 3H7 Fab in white. c Surface representation of CVA6 protomer showing epitopes of 1F4 (light sea green) and 3H7 (orange red). Overlapping regions recognized by both antibodies are highlighted in magenta. d Summary of the epitopes and buried surface areas of the 1F4 and 3H7 MAbs. e Schematic of antibody competition assay by BLI. (Left) No competition: the second CVA6 MAb binds freely to immobilized virions, producing strong signal. (Right) Competition: the first antibody binds to overlapping epitope, blocking the second MAb binding and reducing signal. f, g BLI-based antibody competition assay. Immobilized CVA6-HeB virions were pre-incubated with buffer (reference) or the first antibody, then exposed to the second MAb 1F4 (f) or 3H7 (g). An anti-SARS-CoV-2 MAb served as control (gray curve). Sensorgrams show real-time binding signals of the…

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  Fig. 8. Heparan sulfate serves as the primary attachment receptor for CVA6, whereas KRM1 functions as a post-attachment entry receptor. a Electrostatic surface potential maps of CVA6-HeB viral pentamers. Color scheme: red for negative potential, white for neutral, and blue for positive potential. The black dashed circle highlights the positively charged patch. b Schematic diagram illustrating the sulfation process of heparan sulfate and its role in mediating cellular attachment of multiple enteroviruses (EV). c Wild-type RD cells, ΔKRM1 cells, and ΔSLC35B2 cells were infected with CVA6-HeB, and viral titers were quantified at 48 hpi. Data are means ± SD of triplicate biological samples. p = 0.0422 (ΔKRM1), p = 0.045 (ΔSLC35B2). d CVA6 specifically binds heparin-agarose beads. CVA6-HeB supernatant was loaded onto heparin or control columns, followed by washing and elution. Samples were analyzed by western blotting with anti-CVA6-VP0 antibody. FT, flow through. Two independent experiments were performed, with similar results. e Inhibition of CVA6 attachment to RD cells by soluble heparin. CVA6-HeB was treated with heparin and allowed to attach to cells for 2 h at 4 °C. Attached virus was…