Fig. 5: Reverse genetics to explore the phenotype of a mutant TBEV and molecular dynamic simulations.

a Plaque morphology of mutant (m-rTBEV) and parental (m-TBEV) viruses. b Replication kinetics of both variants in mammalian PS cells and tick cells IRE/CTVM19 (c). d Plaque and RNA content in LoVo-derived immature prM-rTBEV and prM-TBEV after 48 hpi. e, Genomic RNA:PFU ratio for prM-rTBEV versus prM-TBEV. f pH sensitivity of virus samples pretreated with r-furin at acidic or neutral pH, followed by infection of PS cells. g Plaque assay of furin-treated virus samples to assess cleavage efficiency at different pH values. Data in b–g represent at least two independent biological replicates and are shown as mean ± SD. Statistics is done using the Mann–Whitney test; ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. h Schematic of MD simulation setup for (prM-E)₂ dimer in a lipid bilayer. Water molecules and ions are not shown (top panel). i Distributions of per-residue Root Mean Square Fluctuations (RMSF) across all residues of the E dimer. Raw normalized histograms are represented by bars, kernel density estimations as solid lines. j Two dimensional distributions of number of contacts formed between each of the two zippers in the dimer (green) and the neighboring protein E chain (violet), for (prM-E)₂WT (bottom panel, red) and (prM-E)₂MUT (bottom panel, blue) k Distributions of distances between the Cα atoms of residues H208 and G258 in (prM-E)₂, represented as in panel i. l Snapshots of representative configurations of (prM-E)₂WT (S1) and (prM-E)₂MUT (S2-S4). The protein E loop D203-T211 as well as residues L257 and D259 are coloured in white, residues H208 and G258 in gray, and the FCS on prM in red. Black dashed lines indicate the distance between the Cα atoms of residues H208 and G258. m Distributions of distances between the carbonyl oxygen of residue 208 and the amide nitrogen of residue 256 in (sE)₂, represented with red and blue colors indicating (sE)₂WT and (sE)₂MUT, respectively. n Distribution of number of water molecules in the first coordination shell of atoms at the interface of the (sE)₂ dimer, represented as in panel m. Colors indicate WT (red) and mutant (blue) variants throughout panels (i–n).