Fig. 1.

Structure-based engineering of MERS-CoV and SARS-CoV S proteins. (A) Domain architecture of the HCoV-HKU1 S protein and sequence alignment of the helix-turn-helix between heptad repeat 1 (HR1) and the central helix (CH). The two residues colored red are those mutated to proline to retain S2 in the prefusion conformation. FP, fusion peptide; HR2, heptad repeat 2; TM, transmembrane domain. (B) Structure of HCoV-HKU1 S2. Residues shown in sticks in magnified region are those mutated to proline in the 2P variants. (C) Gel-filtration profiles of WT (dashed lines) and 2P-engineered (solid lines) S protein ectodomains from MERS-CoV (blue) and SARS-CoV (red). Each protein was produced from a 1-L transient transfection. All four proteins were expressed with a C-terminal T4 fibritin trimerization domain. The S1/S2 furin site was mutated in MERS S-WT and MERS S-2P. (D) Two-dimensional class averages of negative stained MERS S-WT, MERS S-2P, SARS S-WT, and SARS S-2P. All particles are included. For WT constructs both the prefusion (blue boxes) and postfusion (red boxes) conformations are visible, whereas for the 2P mutants only the prefusion conformation is observed.