Fig. 1
A Ivermectin derivatives used in this study. B Sequence alignment of E proteins from SARS-CoV (1-E), SARS-CoV-2 (2-E), and recent 2-E variants. Boxes indicate the four positions of difference between 1-E (orange) and 2-E, yellow colour indicates amino acid exchanges found in 2-E of new SARS-CoV-2 variants. C Side and top view structural model of the CoV E protein (PDB code: 5 × 29) determined by NMR spectroscopy in lyso-myristoyl phosphatidyl-glycerol micelles [59] The five subunits are colour coded; the N- and C-terminal residues are labelled for one subunit only. D Western blot analysis of 2-E protein and control viroporins expressed in HEK293 cells. All viroporins had an N-terminal myc-tag. Prior to loading, samples were heated briefly to 95 °C to minimize degradation, but multimers were not completely separated. Staining was performed using a primary anti-myc antibody and an AP-conjugated secondary AB. Lane 1: Ladder (ROTI®Mark TRICOLOR: 10, 15, 20, 25, 35, 45, 60, 75); lane 2: GFP; lane 3: SARS-CoV-2 E protein plus signal peptide; lane 4: SARS-CoV-2 E protein without signal peptide; lane 5: SARS-CoV E protein without signal peptide; lane 6: hepatitis C virus p7-1a; lane 7: SARS-CoV-2 ORF3a. Expected molecular weights (kD): SARS-CoV-2 E: 12.6; SARS-CoV-2 E + SP: 16.8, Hepatitis C virus p7-1a: 11.3; SARS-CoV-2 ORF 3a: 35.6 kD Signal peptide (SP): 4.2; SARS-CoV-2 E dimer: 25.2; trimer: 37.8 kD; Hepatitis C virus p7-1a dimer: 22.6; trimer: 33.9. Note immune signal is only observed for myc-tagged antibodies. Small viroporins do not migrate in the same way as globular marker proteins. Suggested oligomers (●, ●●, ●●●) and degradation bands (x) are indicated between lanes 3 and 4 for E proteins, and right of lane 6 and 7 for p7-1a and ORF3a, respectively. E Surface Expression Analysis: HEK293 cells were transfected with cDNA encoding E protein (E), and E protein with membrane-directing signal peptide (E–SP), untransfected and mock-transfected cells were used as control. Left panel: Dot blot. Right Panel: Quantification of Blot intensities, average ± standard deviation from three independent experiments with two spots per treatment (n = 6) is shown. The y-axis break is between 0.001 and 0.02. Background signal (untransfected cells = No DNA) was subtracted from spot intensities; p-values are indicated, **: p < 0.01, ns: not significant, p > 0.05 (one-way ANOVA)