Recombinant Mouse Anti-HEV genotype 1 Antibody (8C11) (CAT#: PABL-130)

Figure 1 Mutational studies on the dimer interface

(A) The schematic representation of wild-type E2 and eleven point mutations targeting the dimer interface region. Secondary structural elements are shown for the E2s region. The mutated residues located on the b-strands and coils are shown in red and black, respectively. (B) These mutants and wild-type E2 were subjected to non-reducing SDS-PAGE and Western Blotting with the neutralizing mAb 8C11 and 8H3 to study the effects of these mutations on dimerization and neutralization, respectively. [+] denotes dimerization or reactivity with 8C11 or 8H3, denotes loss of the respective property. Note that both the capacity to form dimers and the reactivity with mAb 8C11 and 8H3 were abolished simultaneously in six of these mutants: Y557A, T564A, V598E, A599E, L601E and A602E.

Li, S., Tang, X., Seetharaman, J., Yang, C., Gu, Y., Zhang, J.,... & Xia, N. (2009). Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus–host interaction.PLoS pathogens, 5(8), e1000537.

Figure 2 Mutational studies on the groove region.

(A) The schematic representation of wild-type E2 and nine point mutations targeting the solvent-accessible residues near the groove region. (B) The wild type E2 and its mutants were subjected to non-reducing SDS-PAGE and Western Blotting with the HEV-neutralizing antibody 8C11 or 8H3. In this figure the lanes with H indicate samples in the reduced condition (i.e. these samples were heated up to 100uC for 3 minutes). These samples were mainly resolved as monomers. The lanes with N indicate samples in the non-reducing condition (i.e. these samples with 0.1% SDS, no BME and were not heated). These samples were resolved mainly as dimers. All nine mutants remained as dimers. Western Blotting showed that the dimeric E2 wild type and eight mutants were reactive with mAb 8C11. Of these, only E479A, Y485A, I529A, K534A and D496A abolished the 8H3 reactivity. Interestingly, mutant D496A abolished the HEV neutralizing antibodies 8C11 and 8H3 reactivity while maintaining the dimeric arrangement.

Li, S., Tang, X., Seetharaman, J., Yang, C., Gu, Y., Zhang, J.,... & Xia, N. (2009). Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus–host interaction.PLoS pathogens, 5(8), e1000537.

Figure 4 (A and B) The mutants and wild-type E2 were subjected to nonreducing SDS/ PAGE and Western blotting with the neutralizing mAb 8C11 to study the effects of these mutations on E2s(I):8C11 interaction.

The lanes marked with H indicate heated samples in the reduced condition (i.e., these samples were heated up to 100 °C for 3 min) and the lanes marked with N indicate samples in the nonreducing condition (i.e., these samples with 0.1% SDS, no β-mercaptoethanol, and were not heated). (+) Denotes dimerization or reactivity with 8C11, (−) denotes monomer or loss of the respective property.

Tang, X., Yang, C., Gu, Y., Song, C., Zhang, X., Wang, Y.,... & Sivaraman, J. (2011). Structural basis for the neutralization and genotype specificity of hepatitis E virus. Proceedings of the National Academy of Sciences, 108(25), 10266-10271.

Figure 5 (C–E) Sedimentation velocity (SV) was used to detect the mAb 8C11 binding of E2s(I) (C), and the mutants Arg512Ala (D) and Arg512Glu (E).

The c(s) profile of E2, its mutants, or mAb 8C11 alone was denoted as a dashed curve. The profile of the antigen-antibody mixtures was drawn in a solid line. Molar ratio of E2 or its mutant versus mAb 8C11 was 5:1, meaning the antigen was in surplus.

Tang, X., Yang, C., Gu, Y., Song, C., Zhang, X., Wang, Y.,... & Sivaraman, J. (2011). Structural basis for the neutralization and genotype specificity of hepatitis E virus. Proceedings of the National Academy of Sciences, 108(25), 10266-10271.