FIG 4 .

Silencing of ASCC3 restricts viral replication. (A) Expression of ASCC3 mRNA in HeLa cells expressing scrambled or ASCC3 shRNA. The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001). (B to D) Titers of WNV (B), Chikungunya virus (C), and EMCV (D) in the supernatants of HeLa cells transduced with either control or shRNA against ASCC3 at the indicated time points. The results are the averages of three independent experiments performed in triplicate, and asterisks indicate differences from the scrambled shRNA that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05). (E and F) HeLa cells were transfected with expression plasmids encoding GFP or human ASCC3 tagged at the C terminus with HA. (E) The transfection efficiency was measured by flow cytometry using an anti-HA tag antibody and an Alexa Fluor 647-conjugated secondary antibody. (F) Multistep growth analysis of WNV infection in GFP (vector) and human ASCC3-transfected cells. The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01). (G to I) NIH 3T3 cells were transduced with lentivirus carrying a control shRNA or shRNA against murine Ascc3. The mRNA levels of Ascc3 were measured under basal or IFN-β-induced conditions by qRT-PCR and are expressed relative to those after transduction with scrambled shRNA (G). Multistep growth analysis of WNV infection was performed in the corresponding cells without (H) or with (I) IFN-β pretreatment (50 IU/ml for 6 h). The results are the averages of three independent experiments, and asterisks indicate differences that are statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05).