examined the roles of the small Rab5 and Rab7 GTPases, known to be key regulators in vesicular trafficking to early and late endosomes, respectively. To this end, the DN mutants of Rab5 S34N and Rab7 T22N, which have been validated in entry studies with different enveloped viruses, were employed. First, cells were transfected with the control vector pGFP-C1 or constructs expressing GFP-tagged versions of wt and DN form of Rab5 to analyze the transit to early endosomes. After 24 h of transfection cells were infected with DENV-1 or DENV-2 for 1 h at 37uC and then processed to detect GFP expression and internalized viral antigen by indirect immunofluorescence staining of E glycoprotein. When Vero cells were transfected with the wt plasmid, both DENV-1 and DENV-2 virions were internalized in endocytic vesicles exhibiting a speckled and strong virus antigen staining within the cytoplasm and superposition of GFP autoflorescence and viral protein immunofluorescence. In 4 Endocytic Trafficking of Dengue Virus contrast, the expression of the DN affected virus internalization, evidenced by a slight and disperse red fluorescence. To examine the effect of blockade in Rab5-mediated transport on productive infection, transfected cells were allowed to be infected during 24 h before proceeding to cell fixation and staining. The overexpression of Rab5 DN S34N reduced the infection by DENV-1 and the two strains of DENV-2 approximately to 70%, indicating the requirement of the functionality of Rab5 and, consequently, transport of viruses to early endosomes for successful infection. The functionality of Rab5 transgenes in Vero cells was ensured using TRITC-labelled transferrin as control. As expected the overexpression of Rab5 DN S34N reduced the accumulation of transferrin in comparison with cells expressing Rab5 wt, with minor fluorescence intensity in cytoplasm and failure in the superposition of both proteins. Next, the requirement of transport to late endosomes was studied by transfection of Vero cells with GFP-tagged versions of wt and DN mutans of Rab7. The expression of Rab7 DN T22N showed a differential inhibitory effect against DENV strains: it did not affect the infection of Vero cells with DENV-1 HW or DENV2 NGC, but a 65% reduction on viral antigen expression in cells infected with DENV-2 16681 was observed. The functionality of Rab7 transgenes in Vero cells was ensured using JUNV, an arenavirus reported to traffick through Rab7 dependent 5 Endocytic Trafficking of Dengue Virus 6 Endocytic Trafficking of Dengue Virus antibody and TRITC-labelled anti-mouse IgG. B. Cells transfected as in A) were infected with DENV-1 HW, 
DENV-2 NGC or 16681. After 24 h of infection cultures were fixed and immunofluorescence staining was performed as in A. C. For DMXB-A site quantification of samples shown in B, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. D. Cells transfected as in A were then incubated with TRITC-labelled transferrin during 30 min. Then, cells were fixed and fluorescence was visualized. As shown in Fig. 4C, JUNV nucleoprotein expression was diminished in the presence of Rab7 DN T22N while no inhibition of JUNV protein was observed with Rab7 wt. These results demonstrate that the traffic of DENV particles from early to late endosomes should be required for DENV-2 16681 strain whereas the efficient infection with DENV-1 HW and DENV-2 NGC occurs when Rab7mediated transport is block