Figure 7
Endosomal trafficking and low pH dependency is required to mediate infectious entry of DENV into cells. (A) Immunoflourescence analysis reveals co-localization of DENV particles with early endocytic vesicles. Anti-EEA1 was used to stain the early endosomes at 15 min p.i. while DENV particles were stained by anti-DENV E protein antibody conjugated with FITC. (B) By 25 min p.i., majority of the DENV particles are found in association with late endocytic vesicles as shown by confocal imaging. Lysotracker (specific stain for late endosomes) was used to stain the late endosomes within Huh7 cells. Perinuclear distribution pattern of the DENV-associated late endsomes is observed too. (C) Infectious entry of DENV is strongly inhibited in Huh7 cells transfected with dominant negative mutant of Rab5 (DNM) as compared to cells transfected with wild-type Rab5 or mock-transfected cells. (D) Bafilomycin A1 pretreatment of Huh7 cells significantly reduce DENV infection in a dosage-dependent manner. Minimal cytotoxicity is observed for the concentration range of bafilomycin A1 used in this assay. The solvent (DMSO) control was also included in this set of experiment. The average of three independent experiments is shown.