Structural Analysis of the H3 Protein in Monkeypox Virus

(A) Phylogenetic tree depicts the evolutionary relationships of H3 within the Poxviridae family, highlighting MPXV (blue circle), VARV (red circle), and VACV (green circle). (B) Schematic of MPXV adhesion to the cell surface. Viral particles bind to cell surface via specific interaction such as between adhesion protein H3 and HS, followed by membrane fusion mediated by the fusion complex, allowing entry into the host cell. (C) The amino acid sequence of MPXV H3 shows the newly discovered helical structure (240-282, highlight in yellow), the Mg(II) (green) binding site and other potential HS binding motifs (blue underlines). (D) AF2 prediction of MPXV H3 structure. The left and right panels show different orientations of the H3 structure (rotated 180°). The blue region corresponds to H3(1-239), which has a homologous crystal structure (VACV H3, PDB code: 5EJ0). The yellow region represents the AF2-predicted structure of H3(240-282), which remains unresolved in the crystal structure of the homologous protein. All potential GAGs-binding motifs are highlighted with a yellow background. (E) MD simulation snapshot of H3 on a DPPC membrane. H3 is anchored to the membrane through its transmembrane region (residues 283-306, in gray).

Identification of HS Binding Motifs in H3 by MD Simulation and Docking

(A-B) Cartoons show docking results of HS with Motifs 1, 2, 3, and the helical domain, respectively. Panels (B) show RMSD values from 1µs MD simulations, color-coded to match the configurations in (A). (C) Schematics illustrates the reaction coordinate in umbrella sampling, highlighting HS dissociation from H3 with a green force curve. (D) Histogram shows the binding free energies for HS-H3 interactions in motifs 1, 2, 3, and the helical domain. (E) A free energy landscape map from a 1000 ns REMD simulation shows HS binding configurations to the helical and Mg(Ⅱ) regions. (F) Panel provides salt bridge formation statistics between HS and H3’s basic amino acids, with a bar chart of average formations. (G) A surface plot shows the frequency of salt bridge formations within H3, with areas of frequent formations in blue. (H) Detailed views of HS-H3 interactions, with the left image showing salt bridges and the right image displaying electrostatic interactions with Mg(Ⅱ). This panel illustrates the impact on HS binding stability to H3 following the removal of Mg(Ⅱ) during the simulation. (J) The effects of mutating all basic amino acids in the helical domain on the binding stability of HS are shown. (K) The “palm-binding” model is depicted where HS is secured by the helical “fingers” and interacts with the Mg(Ⅱ)-bound “palm.”.

Charge Characteristics and Structural Analysis of H3 Protein

(A) The heatmap shows the amino acid charge distribution in the H3 protein across 66 Poxviridae viruses, following multiple sequence alignment. Blue indicates areas with more positive charges, and red indicates more negative charges. The accompanying curve shows the average charge of all amino acids in Poxviridae H3. (B) Logo plot of the amino acid sequence of the helical region, highlighting the conservation of basic amino acids at specific positions. (C) The surface charge analysis of H3 from the MPXV, VACV, VARV, and cowpox viruses (CPXV) with the helical domain showing a significantly positive charge. (D) Schematic of the single-molecule force spectroscopy unfolding experiment on H3, illustrating the unfolding process of the helical domain (yellow) followed by the main body (blue). (E) Representative curves of H3 unfolding, color-coded to show the helical region (yellow), main body (blue), and full-length (purple) unfolding. (F) Histograms depict the force spectroscopy signals for helical domain, main body, and full-length unfolding, with ΔLc statistics provided. The inset shows a Gaussian fit of unfolding forces.

Analysis of the Helical Domain’s Interaction with HS at Cellular Level

(A) Schematic of the cell force spectroscopy experiment setup shows three scenarios: wild-type H3 on an AFM tip interacting with HS on CHO-K1 cells, mutation of all basic amino acids in the H3 helical region to serine, and cells treated with HS hydrolase to remove surface HS before testing. (B) Force-extension curves depict interactions for the wild type, mutant, and control groups, marked with blue and red asterisks for dissociation events. An inset shows the optical microscope positioning the AFM probe. (C) Histograms of dissociation signals comparing the wild type, mutant, and control groups, with an inset detailing the surface distribution of dissociation forces. (D) Statistical graph showing binding probabilities for different groups, highlighting significant differences determined by t-tests (p<1e-5). (E) FCM results illustrate interactions of wild-type (WT) and Uncharged H3 fused with eGFP with CHO-K1 cells, alongside GFP control (green) and cell-only control (Blank, grey). (F) Statistical analysis of FCM data, showing significant differences between groups as determined by t-tests. *****, P<1e-5

Inhibitor Design Targeting the H3-HS Interaction and Efficacy of AI-PoxBlock723

(A) Diagrams depict protein inhibitors designed to target the H3 helical region, created using RFdiffusion. Sequences capable of folding into the target scaffold structures were generated using ProteinMPNN, and were validated through AF2, followed by 500 ns MD simulations for structural stability and interaction scoring. (B) FCM analysis demonstrates the inhibitory effect of AI-Poxblock723 at various concentrations (0, 10, 50 and 100 μM, x axis). The control group consisted of 2 μM H3-eGFP without the inhibitor, with the relative fluorescence intensity normalized to 1. (C) BLI confirms direct interaction between AI-Poxblock723 and the H3 helical domain. (D) Graphs display the inhibitory effect of the indicated AI-Poxblocks on MPXV infection of Vero E6 cells, with quantitative analysis of virus-infected foci provided on the right. (E-F) BALB/c mice were infected intranasally with 4 × 105 FFU MPXV and treated with single dose of AI-PoxBlock723 (10 mg/kg) or PBS immediately after challenge (n = 5). Infectious MPXV particles and MPXV viral loads in mice lungs at 4 dpi were determined by focus-forming assay (E) and qPCR (F).