RIG-I signaling induces the activation of NF-κB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type I IFNs that restrict further viral propagation. Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52]. Similarly, we show that A20 overexpression in HEK293T cells prevents NF-κB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG-I [53], corresponding to only the two N-terminal CARD domains of RIG-I [RIG-I (2CARD)] (figure 1A, left and middle graph). We next investigated whether A20 also inhibits IRF7 activation. Unlike IRF3, IRF7 is not or weakly expressed under naïve conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation [54], [55]. To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNα4 luciferase reporter construct. RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNα4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7. Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.