Figure 2. The role of Rac1 in DV2 entry.

A. DV2 entry induces inactivation of Rac1 GTPase. ECV304 cells were serum starved for 24 h and infected with DV2. Equal amounts of cell lysates from mock-infected cells, or cells infected with DV2 at different time points as indicated were used to capture the GTP-bound forms of Rac1 GTPase by GST-CRIB pull down assays. The proteins captured were analyzed by SDS–12% PAGE and immunoblotted with anti-Rac1 (top panels). Normalized cell lysates were analyzed for total Rac1 as a loading control (bottom panels). B. Overexpression of dominant-negative Rac1 mutant proteins increases entry of DV2. ECV304 cells were stable transfected with plasmids encoding a wild-type (WT), or two dominant-negative forms (N17, V12N17) of Rac1. The cells were infected with DV2 at 37°C for 1 hour. Then the cells were treated with acid glycine solution (pH 3.0) for 2 min at room temperature to inactivate extracellular viruses, washed twice with PBS, and collected. The titers of cell samples were measured by plaque assay with Vero cells. The titer of Rac1-WT cells was considered as 100%. Experiments were performed in duplicate for at least three independent experiments.