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. Author manuscript; available in PMC: 2019 Feb 8.
Published in final edited form as: Cell. 2018 Jan 25;172(4):797–810.e13. doi: 10.1016/j.cell.2017.12.016

Figure 1. Basal signaling activity of gain-of-function (GOF) variants of MDA5 is mediated by endogenous RNA recognition.

Figure 1

(A) IFNβ reporter activity of wild-type (WT) and GOF MDA5 (R337G, D393V and G495R), with and without premature truncation mutations. Right: a western blot of ectopically expressed, FLAG-tagged MDA5.

(B) Schematic of MDA5 2CARD fused to zinc finger domain (ZF) of Zif268 and DNA oligos containing tandem repeats of the ZF binding site (ZBS).

(C) IFNβ reporter activity of 2CARD-ZF and oligomerization-deficient mutant (m2CARD-ZF, A20K/R21E) in the presence and absence of DNA with 1–4 repeats of ZF-binding sequence (ZBS, GCGTGGGCG) (Jamieson et al., 1996). Right: a western blot of indicated proteins.

(D) Effect of co-expression of dsRNA binding proteins, NS1 and E3L, on the IFNβ reporter activity of G495R (a GOF MDA5 variant) and GST-2CARD. The dsRNA binding-deficient mutants, mNS1 and mE3L, were used as controls. Right: a western blot of indicated proteins.

All cellular assays were performed in 293T cells. Data are represented as mean ± SD (n=3) for (A), (C) and (D). See also Figure S1.