Fig. 2: Identification of possible cholesterol-binding motifs in the M-domain of the ZIKV prM protein.

a A schematic representation of the membrane topology of prM is shown on the left. The trans-membrane segments (TMS), the membrane-associated helix (MH) and the signal sequence are represented as gray and white cylinders, respectively. Proteolytic cleavage sites are indicated by arrowheads specified on the bottom. The pr segment is represented as black circle, M is represented in light gray. Stars indicate locations of identified cholesterol binding motifs (CRAC/CARC). A snapshot from the atomistic molecular dynamics simulations of the membrane-embedded M-domain dimer is shown on the right. The monomers of the dimer are shown in blue and red. The hydrophobic core of the lipid membrane is shown in transparent gray. b Multiple-sequence alignment of M proteins from different flaviviruses using the PRALINE multiple-sequence alignment software. The residues are colored according to their degree of conservation, ranging from 1 (non-conserved) to 10 (100% conserved; *). Potential cholesterol binding motifs are indicated by boxes and their consensus sequences are given on the top. Residues selected for mutagenesis are indicated with black arrowheads. c Experimental approach. Huh7-Lunet T7 cells were transfected with pTM prM-HA constructs for 16 h. Cells were treated for three hours with 5 µM proteasome inhibitor (MG132), followed by a 1-h incubation with 10 µM PAC-cholesterol probe alongside with 5 µM MG132. After crosslinking by UV irradiation, clarified cell lysates were subjected to biotinylation via click chemistry. Lipid–protein complexes were captured using neutravidin-conjugated resin beads and analyzed by western blot. d Representative western blot (n = 5). GAPDH was used as loading control. Molecular weights are indicated on the left (in kilodalton; kDa). e Relative sterol binding of prM proteins was calculated by densitometry of the HA signals that were normalized to input signals. Data are mean ± SEM of the IP:sterol/input signal ratio, normalized to WT. n = 5 independent experiments. One-way ANOVA with Dunnett’s test, **P < 0.01, ***P < 0.001. NS not significant. (WT vs 2-A, P = 0.0058; WT vs 2-S, P = 0.0001; WT vs 3-A, P = 0.0003; WT vs 3-S, P = 0.0006).