Fig 4.

Binding of YFV pr to the virion is enhanced at low pH and prevents sE insertion into membranes at 1:1 stoichiometry. (A) Binding of exogenous pr to YFV 17D viral particle. Western blot with E- or pr-specific antibodies. About 10⁸ ffu of virus was mixed with an excess (1:50) of exogenous purified pr protein and incubated in buffer at different pHs. The complex was then pelleted by ultracentrifugation and analyzed by SDS-PAGE and western blot. The pr doublet is probably the result of the heterogeneous processing of this glycosylated protein. Note that the prM band corresponds to immature virus present in the viral preparation. (B) Histogram representing the values of pr band intensity from two experiments. (C) Co-floatation assay. Five mg of purified sE protein was mixed with different amounts of purified pr protein and with liposomes (refer to Materials and Methods for lipid composition). After addition of buffer at the indicated pH and overnight incubation at 30°C, the protein–liposomes mixture was separated on an Optiprep gradient. Coomassie-stained SDS-PAGE of top (t), medium (m), and bottom (b) fractions is shown. sE protein liposome co-flotation was performed at pH 8 (left columns) and at pH 6 in the presence of pr at sE:pr molar ratios 0.3, 1, and 3 (right columns). (D) Histogram of normalized sE band intensity from top and bottom fractions to the amount of sE present in the bottom fraction at pH 8. Several flotation assays were included in the calculation using ImageJ software. Errors are standard deviations calculated from at least two experiments.