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. 2020 Apr 15;11:656. doi: 10.3389/fmicb.2020.00656

FIGURE 1.

FIGURE 1

HEV ORF2 protein inhibits IFN-β promoter activation by downregulating the RIG-I pathway. (A) Plasmids expressing HEV-1 proteins were transfected into the HEK293T cells (25 ng) along with RIG-I (0.5 ng) and IFN-β firefly and TK Renilla luciferase reporters. RIG-I was induced with 3pdsR27 24 h post-transfection. Luciferase assay was done 16 h post-induction. (B) Plasmids expressing HEV-3 proteins were transfected into the HEK293T cells (25 ng) along with RIG-I (0.5 ng) and IFN-β firefly and TK Renilla luciferase reporters. RIG-I was induced with 3pdsR27 24 h post-transfection. Luciferase assay was done 16 h post-induction. (C) For IPS-1 assay, 0.5 ng of IPS-1 was co-transfected with two different quantities of HEV-1 ORF2, HEV-3 ORF2 plasmids (10 and 25 ng) or 10 ng of HEV-1 ORF3 plasmid along with IFN-β firefly and TK Renilla luciferase reporter plasmids. Luciferase activity was measured 24 h post-induction. (D) HEK293T cells were co-transfected with 0.5 ng of IPS-1 plasmid, HEV-1 ORF2 or ORF3 plasmids (25 ng each) individually or in combination along with IFN-β firefly and TK Renilla luciferase reporters. Luciferase activity was measured 24 h post-transfection. (E) 0.5 ng of WT RIG-I or RIG-I K858E plasmid was co-transfected with 25 ng of HEV-1 ORF2 plasmid or ORF3 plasmid along with IFN-β firefly and TK Renilla luciferase reporter plasmids. Induction was given 24 h post-transfection with 40 HAU/ml of Sendai virus (SeV). Luciferase activity was measured 16 h post-induction. (F) Effect of the HEV-1 ORF2 and ORF3 on NF-κB activity. RIG-I assay was performed by co-transfecting 0.5 ng of the RIG-I plasmid, the HEV-1 ORF2 or the HEV-1 ORF3 plasmids (25 ng) along with the NF-κB firefly and TK Renilla luciferase reporter plasmids. SeV infection was given 24 h post-transfection. Luciferase activity was measured 16 h post-induction. All experiments were performed in the HEK293T cells. Values are mean ± SD, n = 3 (∗∗∗ denotes p-values ≤ 0.001, ∗∗ denotes p-values ≤ 0.01, and ∗ denotes p-values ≤ 0.05).