Fig 2: S-Acylation controls the lipid environment of spikeSee also Figure S4.(A) Coarse-grained (CG) model of spike’s transmembrane (TM) helix + C-terminal region (residues 1,201-end in UniProt P0DTC2) in trimeric form, inserted into a DPPC-(yellow)-DLiPC-(red)-cholesterol-(green) membrane model. Left, starting conditions: random lipid positions and stretched cytoplasmic tail; center, representative end state of same view after 3.75 μs of CG MD simulation of the S-palmitoylated protein depicts phase separation and spike partitioning. Right, side view of the same model after 2 μs of atomistic MD simulation in the S-palmitoylated form in a POPC membrane.(B) Quantification of contacts at equilibrium between the full TM helix, the luminal half, or the cytosolic half, and each lipid phase. Results are means ± SD (n = 10).(C) Vero E6 expressing spike-HA WT surface labeled with biotin and processed for fractionation. Western blot (WB) of biotinylated proteins (Surface-Strp. Pull-down) precipitated from each fraction were compared to unbound fractions (intracellular-unbound).(D) Quantification of spike-HA (all forms) in each fraction relative to the total signal in all 6 fractions. Results are mean ± SD, n = 3.(E and F) High-throughput automated microscopy of Vero E6 expressing spike-HA (WT, or 10C-A mutant) for 24 h immunolabeled for HA and stained with Filipin. (E) Total levels of filipin staining within HA-positive cells (or per cell—mock sample) quantified per number of nuclei. The data were averaged for 49 frames over 8 wells per condition. Each dot represents one independent well, and results are mean ± SEM. Equivalent results were obtained for three independent assays.(G) Vero E6 infected with SARS-CoV-2 24 h, MOI ≈ 0.1 were incubated with beta-methylcyclodextrin (βMCD) or not (Ctrl) for 30 min. Cell extracts were fractionated as in (C). DRM marker Caveolin1 (Cav1)-positive control, transferrin receptor (TfR)-negative control.(H and I) Quantification of spike forms (H) and caveolin 1 (I) signals as in (C). Results are mean ± SD, n = 3.(J) Vero E6 transfected with indicated siRNA (siCtrl or siMixT pool), infected and fractionated as in (G).(K–M) Quantification of (K) and (L). Spike (all forms) and (M) ERGIC53 in each fraction as in (C). Results are mean ± SD, n = 3. All p values were obtained by two-way (except for E, one-way) ANOVA.