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. 2019 Jul 30;93(16):e00211-19. doi: 10.1128/JVI.00211-19

FIG 2.

FIG 2

Functional validation of the ZIKV CRISPR activation screen. (A) Huh7 cell lines expressing the indicated sgRNAs were infected with ZIKV (MOI, 0.2) for 2 h. After 7 days, the cells were stained with crystal violet. The graph shows the quantification of the crystal violet staining by measurement of the absorbance at 570 nm. Bar graphs show mean values ± standard deviation (SD) from two independent experiments performed in duplicate. ****, P < 0.001, one-way analysis of variance (ANOVA), Dunnett’s multiple-comparison test. (B) Expression levels of the activated genes as determined by real-time PCR using gene-specific primers. Shown are mean values ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, multiple two-tailed unpaired t tests. (C) Representative crystal violet staining results. (D) Time course of relative ZIKV RNA levels as determined by real-time PCR following infection (MOI, 1) of nontargeting (NT), IFI6, and IFN-λ2 CRISPR activated cell lines. ****, P < 0.001, two-way ANOVA with Tukey’s multiple-comparison test. (E) Time course of ZIKV production NT, IFI6, and IFN-λ2 CRISPR activated cell lines as determined using plaque assays. ****, P < 0.001, two-way ANOVA with Tukey’s multiple-comparison test. (F) Flow cytometry of infected Huh7 cells expressing NT or IFI6 sgRNAs at 48 h postinfection. The results are the mean values ± SD. *, P < 0.05, two-tailed unpaired t test. (G) ISG56-luciferase activity in cells transfected with MAVS, empty FLAG plasmid, or an IFI6-FLAG expressing plasmid. The results are the mean values ± SD. n.s., nonsignificant, two-tailed unpaired t test. (H) IFI6 mRNA levels in cells transfected with IFI6 or nontargeting control siRNAs in the presence or absence of IFN-α (1,000 U/ml, 18 h), **, P < 0.01, two-tailed unpaired t test. (I) IFI6 knockout slightly rescues ZIKV infection in IFN-α-treated cells. Huh7 cells were transected with the indicated siRNAs. The next day, the indicated samples were pretreated with IFN-α (for 2 h) and infected with ZIKV (MOI, 1). The cells were harvested for qPCR analysis at 24 h postinfection. *, P < 0.05, two-tailed unpaired t test.