Fig. 7: M1R-01 recognizes the conserved epitopes across orthopoxvirus and plays a neutralizing role in the post-binding stage.

a Alignment of M1R extracellular domain in 11 viral strains within Orthopoxvirus. Different colors indicate the degree of conservation at different sites: clear is completely consistent, followed by light blue, and the site with the greatest difference is represented by the deeper blue. Above the sequence, the secondary structures, such as α helices and β sheets that correspond to the amino acid positions, are represented. The red dots indicate the key residues on the M1R protein that are associated with the binding to M1R-01 (corresponding to the results in b). b Pull-down assay to determine the critical residues in M1R for association with Fc-M1R-01. The key sites are labeled in blue. c Co-IP assay to determine the key amino acids in M1R-01 for association with the antigen M1R. d Schematic diagram of experimental determination of the virus entry steps affected by M1R-01. For the binding step, the VACV was mixed with Tri-M1R-01, added into the pre-cooled cells, and allowed for incubation at 4 °C for 1 h. Then, the cells were washed with cold PBS and harvested for qRT-PCR analysis of viral DNA copies. For the post-binding step, the VACV was first incubated with the target cells at 4 °C for 1 h, and then moved to 37 °C. Tri-M1R-01 or control isotype Nb was added at different timepoints, and plaques were counted at 48 h post-infection. e Quantification of the cell-bound virus in the presence or absence of antibodies. Mock means cells without the addition of VACV and antibodies. Isotype Nb is a non-relevant trivalent Nb as the negative control. Bars indicate mean ± SEM of triplications. f Inhibition of VACV plaque formation by antibodies added at different post-binding timepoints. Bars indicate mean ± SEM of triplications. t-test was used for comparisons between groups. Statistical significance was shown as *P < 0.05, **P < 0.01, ***P < 0.001.