Fig. 3.

The role of VCP in distinct steps of WNV life cycle was investigated. (A) WNV-VLP infection after the indicated siRNA treatment, HeLa cells were treated with siRNA against VCP, siVCP (1) or control siRNA (siCont). The indicated siRNA treated-cells were inoculated with WNV-VLPs (16 HAU) at 48 h post transfection and incubated for 72 h. Relative quantification of WNV-RNA normalized to human β-actin from (A) examined by qRT-PCR. Mean ± SD from three independent experiments is shown; ** p < 0.01 (one-way ANOVA). (B) Relative quantification of WNV-RNA expression levels normalized to human β-actin of siRNA-treated cells inoculated with WNV at the early time point of infection. siRNA-treated HeLa cells were inoculated with WNV after 48 h post siRNA transfection. The inoculated cells were incubated on ice for 1 h, followed by washing 5 times with PBS and then transferred to 37 °C. After 1 h incubation, the cells were harvested by trypsin and prepared for qRT-PCR. Mean ± SD from two independent experiments in triplicate is shown; * p < 0.05 (one-way ANOVA). (C) HeLa cells were treated with siRNA against VCP, siVCP (1) or control siRNA (siCont). After 24 h post transfection, the cells were transfected with plasmid set for WNV-VLP production and incubated for 72 h. The culture supernatants were harvested and inoculated on Vero cell monolayers in 10-fold serial dilutions. The viral titers of the harvested supernatants were determined as IFU/ml. Mean ± SD from three independent experiments is shown; ** p < 0.01 (one-way ANOVA). (D) HeLa cells were treated with siRNA, either siVCP (1) or siCont, and then transfected with plasmid containing WNV DNA replicon, pCMV-WNrep-DsRed at 24 h post siRNA treatment and incubated. The transfected cells were harvested at 72 h post transfection of pCMV-WNrep-DsRed. Relative quantification of WNV-RNA normalized to expression of human β-actin was examined by qRT-PCR. Mean ± SD from three independent experiments is shown; ** p < 0.01 (one-way ANOVA).