Endocytic pathway followed by dengue virus to infect the mosquito cell line C6/36 HT

The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.