Fig. 3. The cytoplasmic tail of the SARS-CoV-2 S protein harbours di-acidic ER export motifs.

a C-terminal sequence of the D/E to A mutant of S indicating the putative di-acidic ER export motifs mutated to alanine. b Micrographs of U2OS cells transiently expressing N-terminally HA-tagged wild-type S and the D/E to A mutant. Cell surface S was initially stained under non-permeabilising conditions with an anti-HA antibody labelled with Alexa Fluor (AF) 647. Cells were then permeabilised and stained with anti-HA labelled with AF488 to detect the internal S. Scale bars 10 μm, with the images for the individual channels taken at the same magnification as the merged image that has the scale bar. The experiment was repeated twice. c Quantification of (b) by flow cytometry. Displayed are overlaid histograms representing the ratio of extracellular S (AF488 signal) to that of intracellular S (AF647 signal) for wild-type S and the D/E to A mutant. Histograms normalised to the mode value, and represent >10,000 events. Chi-squared test shows the difference in the median ratios (wild-type, 0.78; D/E > A, 0,096) to be statistically significant (P = 0.01, 99% confidence). Representative of six independent experiments.