Figure 5. Covalently linked sE dimers recapitulate the EDE and do not interact with liposomes.

(a,b) ELISA plates were coated with DENV2 either wild type monomeric sE (sE WT) or the two covalently linked sE dimers (a) A259C or (b) L107C/A313C and following incubation with panel of anti-EDE1 and anti-EDE2 mAbs (left panel) or anti-FLE at 1 μg ml⁻¹ (right panel), binding was determined using ALP-conjugated anti-human IgG. (c) Results of co-flotation with liposomes in an Optiprep gradient at low pH (see Methods for lipid composition). Wild type, single and double mutants from DENV2 FGA02, DENV3 H86 and DENV4 1-0093 were incubated at pH 5.8 with liposomes and run in an Optiprep gradient. Insertion of the WT sE proteins in the liposome membrane results in its floatation to the low density top (T) fractions of the gradient (lanes 1). A fraction of the single mutants appears to still be able to float with the liposomes (lanes 3), whereas in the double mutants, there is no sE protein recovered from the top fraction (lanes 5), in line with the fact that the FLE is not exposed.