Structure and design of Langya virus glycoprotein antigens

Significance Langya virus (LayV) was discovered in febrile patients in China for which no countermeasures exist. We describe the architecture and antigenicity of the LayV fusion (F) and attachment (G) glycoproteins, suggesting that vaccines and therapeutics currently being developed against Nipah virus (NiV)/Hendra virus (HeV) will be ineffective for LayV. We designed stabilized version of each glycoprotein to support the development of vaccines and therapeutics against these pathogens.


Fig. S5
Fig. S5 CryoEM data processing pipeline for GhV F. A-B, Representative electron micrograph (A) and 2D class averages (B) of GhV F embedded in vitreous ice.The scale bar represents 100 nm (A) or 150 Å (B).C, Gold-standard Fourier shell correlation curve for the GhV F reconstruction.The 0.143 cutoff is indicated with an horizontal blue line.D, Data processing flowchart.CTF: contrast transfer function; NUR: non-uniform refinement; Polishing: Bayesian particle polishing implemented in Relion.E, Sharpened GhV F reconstruction colored according to local resolution calculated using cryoSPARC.

Fig. S6
Fig. S6 Incompatibility of the prefusion LayV F structure with binding of two NiV/HeV F neutralizing mAbs.A, Superimposition of the 5B3-bound NiV F structure (PDB 6TYS(18)) onto prefusion LayV F. Two LayV F protomers are shown in blue and red whereas the 5B3 heavy and light chains are rendered gold and yellow respectively.NiV F is omitted for clarity.N-linked glycans are shown as green surfaces.Red circles indicate potential clashes.B, Superimposition of the 12B2-bound HeV structure (PDB 7KI4(17)) onto prefusion LayV F with the same color and representation scheme as in panel A.

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Fig. S7 CryoEM data processing pipeline for postfusion LayV F in complex with the 4G5 Fab.A-B, Representative electron micrograph (A) and 2D class averages (B) of 4G5-bound postfusion LayV F embedded in vitreous ice.The scale bar represents 100 nm (A) or 200 Å (B).C, CryoEM data processing flow chart including local resolution maps computed using cryoSPARC.The masks used for local refinement are shown in pink and purple.Homo Refine: homogeneous refinement.CTF: contrast transfer function; NUR: non-uniform refinement; Polish: Bayesian particle polishing implemented in Relion.

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Fig. S8 Characterization of MojV and LayV F/G cross-reactive antibodies.A, Biolayer interferometry binding analysis of 100 nM of LayV F (red) or NiV F (orange) to the 4G5 IgG immobilized at the surface of AMC biosensors showing that 4G5 recognizes LayV F but not NiV F. B-C, Assessment of cell-cell fusion mediated by LayV F/G (B) or MojV F/G (C) in presence of varying concentrations of the 4G5, 2B2 or D61 (anti HIV gp41, NC:

Fig. S9
Fig. S9 Characterization of designed LayV G mutants.A, Electron micrographs of negatively stained LayV G mutants after affinity purification and prior to size exclusion chromatography.B, 2D class averages obtained from negatively stained LayV G mutants showing the formation of tetramers for all mutants evaluated.Template picking and a prior round of 2D classification was used to enrich well-folded particles.C, Size exclusion chromatography profiles of LayV G mutants highlighting variations in protein aggregation and tetramerization.

Fig. S10
Fig. S10 CryoEM data processing pipeline for LayV GSM harboring the oP4h Nterminal fusion A-B, Representative electron micrograph (A) and 2D class averages (B) of LayV GSM harboring the oP4h N-terminal fusion embedded in vitreous ice.The scale bars represent 100 nm.C, Data processing flowchart.CTF: contrast transfer function; NUR: non-uniform refinement.D, 3D maps corresponding to 3D classifications with and without alignment referenced in the data processing flow chart.Classes highlighted in red were used for further data processing.E, Gold-standard Fourier shell correlation curve for the LayV G reconstruction.The 0.143 cutoff is indicated by the blue line.F, Local resolution map calculated using CryoSPARC and plotted onto the sharpened LayV G reconstruction.

Fig. S12
Fig. S12 Stalk stabilizing mutations and comparison of the stalk sequence with other HNV G glycoproteins.A, Designed LayV G stabilizing mutations (labeled white spheres) in the stalk of the LayV GSM cryoEM structure highlighting pi-helices (yellow), and a 310-helix (orange).The head domains are shown as black surfaces.B, Zoomed-in views of stabilizing mutations with relevant and neighboring side chains rendered as sticks colored as in (A).C, Amino acid sequence alignment spanning LayV G residues 79-143 with other HNV G glycoproteins.Known or predicted residues facing the interior of the tetrameric coiled coil are shown in beige.Regions with known or predicted pi-helical structures are highlighted in yellow.The region with known or predicted 310-helical structure is highlighted in orange.K85 is marked with an asterisk.Residues mutated for stability are indicated with a dagger.

Fig. S13 .
Fig. S13.CryoEM data processing pipeline for LayV GSM harboring the K85L/L86K mutations without oP4h fusion.A-B, Representative electron micrograph (A) and 2D class averages (B) of LayV GSM harboring the K85L/L86K mutations without oP4h fusion embedded in vitreous ice.C, Data processing flowchart.CTF: contrast transfer function; NUR: non-uniform refinement.D, Gold-standard Fourier shell correlation curve for the LayV G reconstruction.The 0.143 cutoff is indicated by the blue line.F, Local resolution map calculated using CryoSPARC and plotted onto the sharpened LayV G reconstruction.