Fig 1. Clathrin is not required for DENV-3 infective internalization into Vero cells.

(A) Cells were treated with chlorpromazine or dansylcadaverine and infected with DENV-3. After 1h of internalization in presence of the drugs, monolayers were treated with proteinase K and the cell pellets were plated onto Vero cells to determine internalized virus by an infectious centre assay. (B) Cells treated with 40 μM chlorpromazine, 150 μM dansylcadaverine or untreated (control) were infected with DENV-3. At 48 h p.i., immunofluorescence staining was carried out using mouse anti-E glycoprotein antibody. (C) Cells transiently transfected with GFP-DIII∆2 or GFP-EH29 were infected with DENV-3. After 24 h cells were fixed and viral antigen expression was visualized by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG. (D) For quantification of samples shown in C, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. (E) Vero cells were infected and processed as in (A) to obtain cell pellets, then total RNA was extracted and real-time RT-PCR was performed to determine the amount of internalized viral RNA molecules. In (A), (D) and (E) results are expressed as the mean of three independent experiments ± SD. Asterisks indicate statistical significance (*** p < 0.001), ns: non-significant difference between treated sample and control.