WNV NS1 and NS5 are responsible for <i>Cx</i>Cul4 regulation.

<p><b>A.</b> Hsu cells were transfected with WNV genes (C-Capsid; NS1, NS3, NS4A or NS5). Total RNA was collected at 24 hours post-transfection and real time RT-qPCR was performed using <i>Culex</i> Cul4-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, significant from empty vector control). <b>B.</b> Real time Rt-qPCr was performed on cells treated as in <b>A</b>, using <i>Culex</i> Cullin3-specific primers. <b>C</b>. Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). GFP dsRNA was used as a silencing control (Control). At 24 h post-transfection, the cells were infected with WNV and cell lysates were collected 24 hours post-infection. Western blot was performed using anti-<i>Culex</i> STAT and anti-beta actin antibodies, with marker representing proteins with known molecular weight (in kDa). The experiment was repeated 3 times and representative blots shown here. <b>D</b>. Hsu cells were transfected with WNV genes (NS1, NS5 or NS3) along with dsRNA against <i>Culex</i> cullin (+). GFP dsRNA was used as a silencing control (-). Control indicates empty vector transfection. Cell lysates were collected 24 hours post-infection. Western blot was performed using anti-<i>Culex</i> STAT and anti-beta actin antibodies. The experiment was repeated and representative blots shown here. <b>E.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). GFP dsRNA was used as a silencing control (GFP dsRNA). At 24 h post-transfection, the cells were infected with WNV and treated with MG132 (1 μM) and cell lysates were collected 24 hours post-infection. Immunoprecipitation was performed using anti-ubiquitin antibody, followed by Western blot using anti-ubiquitin (Upper) and anti-<i>Cx</i>STAT (lower) antibodies. WCL represents whole cells lysate (pre-IP), FT represents flow-through and IP represents immunoprecipitate (eluate). The experiment was repeated twice and representative blots shown here. <b>F.</b> Female <i>Culex annulirostris</i> mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV for 1h. At 2 days and 10 days post-infection, mosquitoes were collected and processed for Western blot using anti-<i>Cx</i>STAT and anti-actin antibodies. The experiment was repeated and representative blots shown here.