Fig. 5.

CTD deletion of both RIG-I and MDA5 locked them in an auto-repressed form which is alleviated by deletion of the pincer domain in the case of RIG-I. a CARD-hel, RIG-I 1–797 and MDA5 1–895, and CARD-hel° constructs and their DQCH mutant display only background activity similar to their full-length (wt) counterparts and do not respond to poly(I:C). Note by comparison the significant albeit reduced response to poly(I:C) of full-length RIG-I and MDA5 with CTD° unable to bind RNA (P <0.025 for both) when compared to CTD° without poly(I:C), and P <0.0025 and P <0.0125 when compared to wt constructs + poly(I:C), respectively, consistently with data shown in Figs. 2 and 3. b Further deletion of the pincer domain alone, RIG-I 1–744 quoted Δ[pincer-CTD], or together with deletion of the last hel2 βsheet, RIG-I 1–734 quoted Δ[β13pincer-CTD], results in constitutive activity of RIG-I (P <0.025 and below). In contrast, their MDA5 counterparts MDA5 1–836 quoted Δ[pincer-CTD] and MDA5 1–824 quoted Δ[β13pincer-CTD], shows no enhancement over the background activity exhibited by MDA5 hel and hel° constructs (P >0.05 and above). Free CARDs from both RIG-I (aa 1–228) and MDA5 (aa 1–294) are constitutively active (P <0.01 for both). Note that the lower basal activity (*) of MDA5 Δ[pincer-CTD] construct is associated with a lower expression level. CARD: caspase activation and recruitment domain; CTD: C-terminal domain; hel: helicase