Figure 4. BMP, a lipid specific to late endosomes, is required for nucleocapsid delivery into the cytoplasm and viral infectivity but not membrane fusion.

Cells were cultured overnight in media containing 50 µg/ml anti-BMP antibody. Cells were washed and the medium was changed before immunostaining, treatment with R18-labeled JE-VLPs (200 µl at 17 pM) or with YFV (MOI = 0.1–1). (A) Immunostaining of BMP in BHK cells. Left: BMP staining (Texas Red) was mainly perinuclear. Center: NS1-GFP (green) expression in BHK cells to transcomplement the ΔNS1-YFV genome with NS1 for viral production as described in Ref. [37]. (B) BHK cells treated with total mouse IgG instead of anti-BMP antibody. No antibody staining is detectable in the cells (left panel). JE-VLPs, (C), or YFV, (D), fused normally in Vero cells pretreated with anti-BMP antibody, as indicated by the R18 fluorescence dequenching profiles of three representative tracked particles. Error bars represent the standard error of the mean (SEM) of three experiments. (E) qRT-PCR assay showing that pretreatment of Vero cells with anti-BMP antibody reduced the delivery of YFV nucleocapsid into the cytoplasm by approximately 70% relative to untreated cells or cell treated with total mouse IgG. (F) Plaque assay showing that pretreatment of BHK cells with the anti-BMP antibody partially inhibited YFV replication, reducing the number of viral plaques by approximately 65% relative to the controls.