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. 2021 Aug 15;10(1):1609–1625. doi: 10.1080/22221751.2021.1964384

Figure 2.

Figure 2.

ZIKV actively antagonized interferon signalling. Coinfection with ZIKV abrogated the IFN-induced reduction in VSV infection in Huh7.5 cells (a) and JEG-3 cells (b). (a) Huh7.5 cells were infected with ZIKV (MOI = 1) for 24 h and then treated with IFN-α (2000 U/ml) for 8 h, followed by infection with VSV (MOI = 0.05) for 28 h. The levels of VSV genomic RNA in the harvested cells were determined by qRT-PCR and normalized to the level of GAPDH RNA. The mean ± SD of three biological replicates is shown (n = 3). (b) JEG-3 cells were infected with ZIKV (MOI = 1) for 24 h and then treated with IFN-α (2000 U/ml) for 8 h, followed by infection with VSV (MOI = 0.05) for 16 h. The levels of VSV genomic RNA in the harvested cells were determined by qRT-PCR and normalized to the level of GAPDH RNA. The mean ± SD of three biological replicates is shown (n = 3). (c) western blotting analysis of the Huh7.5 cell line expressing the ISRE-HA-RFP reporter. Cells were treated (+) or not (−) with IFN-α (2000 U/ml) for 24 h and then analysed by western blotting with the indicated antibodies. A representative picture of multiple independent experiments is shown. (d–h) ZIKV antagonized ISRE-dependent IFN signalling. (d) The upper panel shows the experimental design for c–f. Huh7.5-ISRE-HA-RFP cells were infected (+) or not (−) with C7 or C7.D29 at an MOI of 1 or 10 for 48 h and then treated (+) or not (−) with IFN-α (2000 U/ml) for 24 h. The harvested cells were analysed by western blotting with the indicated antibodies. Representative blots of three biological replicates are shown. The values to the left of the blots are molecular sizes in kilodaltons. (e–g) The protein abundances of each protein in C were quantified and plotted. The mean ± SD of three biological replicates is shown (n = 3). (h) ZIKV RNA levels were quantified by quantitative RT-PCR and normalized against GAPDH RNA levels. The mean ± SD of three biological replicates is shown (n = 3). Statistical analysis was performed between C7- or C7.D29-infected groups and uninfected groups (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed, unpaired t-test).