Fig 8.

The GTPase Rho A is required for JEV infection of neuronal cells. (A to C) Neuro2a cells were transfected with myc-tagged constructs of wt RhoA, RhoA L63, RhoA N19, Rac1 L61, Rac1 N17, wt Cdc42, Cdc42 L61, and Cdc42 N17. The GTPase activity of the overexpressed constructs was quantified using GTPase-specific ELISA as described in Materials and Methods. (D) Neuro2a cells transfected with the wt, DA, and DN constructs of Rho, Rac, and Cdc42 were infected with JEV (MOI, 1). At 24 hpi, double immunofluorescence staining was done for myc and JEV E. Cells staining positive for both myc and JEV E were scored and normalized to wt RhoA-expressing infected cells. (E) Time course of Rho activation in response to JEV binding using Rho GTPase-specific ELISA as described in Materials and Methods. (F) Neuro2a cells were serum starved for 2 h before treatment with CT04 inhibitor for another 2 h. Cells were infected with JEV (MOI, 0.4) in the presence of inhibitor. Infection was quantified as described in Materials and Methods. (G) Virus endocytosis (MOI, 10) was quantified in control and CT04-treated cells by qRT-PCR. Student's t test was used to generate P values, **, P < 0.01; *, P < 0.05.