Figure 6.

USP18 expression displaces NS5 from STAT2 and overcomes ISG15 deficiency. (A) Flag-tag immunoprecipitation (IP) assay and Western blot analysis of HEK293 ISG15 KO cells transfected with ZIKV NS5-FLAG, human USP18 WT, human USP18 C64A mutant or the empty vector (pcDNA3.1) plasmids, followed by IFNα2b (100 IU/ml) priming for 18 h. WCL, whole cell lysate. Results are representative of three independent experiments. (B) STAT2(V5) IP assay and Western blot analysis of HEK293 ISG15 KO cells transfected with the indicated plasmids, followed by IFNAα2b (100 IU/ml) priming for 18 h. Results are representative of three independent experiments. (C, D) Complementation with USP18 in A549 ISG15 KO cells. Cells were stably transfected with human USP18 or the empty vector and infected with DV at an MOI of 0.01. At 36 hpi, cells were harvested and cell lysates were analyzed by Western blot with the corresponding antibodies (C). Percentage of cells infected as measured by E protein staining (D). Error bars represent mean ± SD. Results are representative of two independent experiments. Statistical analyses were conducted using Mann-Whitney’s test in Prism 8 (GraphPad Software). p value **<0.01 (D). EV, empty vector.