FIG 4.

The de novo formation of DENV replication organelles is not impacted by NPP3C or 6A1HI treatment. (A) Schematic representation of DENV genome and pIRO-D plasmid. NTR: nontranslated region; CS: cyclization sequence; EMCV IRES: Internal ribosome entry site of encephalomyocarditis virus. (B–F) Huh7/Lunet-T7 cells were transfected with pIRO-D plasmid and treated for 12 h with DMSO, 50 μM NPP3C, or 50 μM 6A1HI. 16 h posttransfection, cells were analyzed for immunofluorescence, western blot and electron microscopy. (B) Transfection efficiency upon treatment with the different compounds was evaluated by immunostaining with anti-NS3 antibody. (C) Total lysates of transfected and treated cells were analyzed by western blot using the indicated antibodies. (D) Electron microscopy analysis of samples treated as in (A). Scale bar: 500 nm (E) Quantification of the number of cells showing vesicle packets. At least 15 cells from two different experiments have been analyzed for each condition. (F) Measurement of VP diameter.