FIG 1.

NS1-induced cell activation promotes lipid raft accumulation on the cell membrane and facilitates DENV attachment. (A) RAW 264.7 cells were incubated with 50 μg/ml NS1 for up to 24 h. Lipid rafts were quantified by flow cytometry using CTB-FITC, and the mean fluorescence intensity (MFI) was calculated relative to control cells incubated with PBS. (B) Immune activation was assessed by quantification of secreted NO in culture supernatants. (C) Total cholesterol was quantified in cell extracts from NS1-treated cells. (D) MHC-II expression on the cell surface was assessed by flow cytometry of NS1-treated cells. (E) Cells were treated with NS1 for 24 h in the presence of the LPS inhibitor PMB (100 μg/ml) or the LPS competitor LPS-RS (100 μg/ml). Lipid rafts were assessed by CTB -FITC incorporation. (F) Secreted NO was quantified in the supernatants by the Griess assay. (G) NS1-treated cells were challenged with DENV2 for 1 h at 4°C for viral attachment. Unbound virus was washed out with PBS, and total RNA was extracted. vRNA was quantified by qRT-PCR. Error bars represent the SDs of at least three biological replicates. Asterisks represent significant differences, compared to control. *, P < 0.1; ***, P < 0.001; ns, not significant.