Fig. 4.

Property alteration of native HEV while binding with 8C11. (A) Assumed binding modality of 8C11 and 3B6 to native HEV (proposed T = 3) illustrated according to the cocrystal structure of E2s:8C11 and the cryo-EM structures of VLP:3B6 and T = 3 HEV VLP. One E2s monomer is colored green, and the models of binding 8C11 and 3B6 Fabs are colored blue and red, respectively. (B) Virus pelleting assay of native HEV pretreated with 8C11 and 3B6. Native HEV pretreated with 8C11 or 3B6 or alone were pelleted by ultracentrifugation to quantify copy numbers of the RNA genome. Input column in the histogram indicates the RNA genomic copy of native HEV before ultracentrifugation. Results were statistically analyzed by an unpaired Student’s t test. *P ≤ 0.05; ns, not significant (8C11 vs. 3B6, P < 0.0001; 8C11 vs. control, P < 0.001; 3B6 vs. control, P = 0.6752 > 0.05). The decrease in RNA copy number in the 8C11-treated sample as compared with the control suggests that half of the native HEV changes to a smaller material that cannot be pelleted as an intact virus. (C) Real-time PaSTRy assay of native HEV pretreated with 8C11 and 3B6. There is a trend for an increase in fluorescence from the dye bound to the HEV RNA genome exposed by 8C11 binding; this indicates that the HEV capsid is subjected to ongoing perturbation with 8C11 treatment over ∼60 min. The fluorescent signal reaches a plateau early (∼60 min) and remains there until the assay end point (∼150 min). As for the controls, there were no substantial changes in the fluorescence signals of 3B6 or the no-antibody control.