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[Preprint]. 2020 Oct 27:2020.10.26.356048. [Version 1] doi: 10.1101/2020.10.26.356048

Figure 2. MDA5 activation requires ISGylation at K23 and K43.

Figure 2.

(a) Endogenous MDA5 ISGylation in NHLFs that were mock-treated, transfected with HMW-poly (I:C) (0.1 μg/mL) for 40 h (left), or infected with DENV or ZIKV (MOI 1 for each) for 48 h (right), determined by immunoprecipitation (IP) with anti-MDA5 (or an IgG isotype control) followed by IB with anti-ISG15 and anti-MDA5. WCLs were probed by IB with anti-ISG15 and anti-Actin (loading control). (b) ISGylation of FLAG-tagged MDA5–2CARD and MDA5ΔCARD in transiently transfected HEK293T cells that also expressed V5-ISG15, HA-Ube1L, and FLAG-UbcH8, assessed by FLAG pulldown (PD) and IB with anti-V5 and anti-FLAG forty hours after transfection. WCLs were probed by IB with anti-HA, anti-FLAG, anti-V5, and anti-Actin. (c) Endogenous MDA5 ISGylation in ISG15 KO HeLa cells stably reconstituted with vector, WT ISG15 or ISG15-AA and co-transfected with HA-Ube1L and FLAG-UbcH8 after IFN-β treatment (1,000 U/mL) for 24 h, determined by IP with anti-MDA5 and IB with anti-ISG15 and anti-MDA5. (d) ISGylation of GST-MDA5–2CARD WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8 for 24 h, determined by GST-PD and IB with anti-V5 and anti-GST. (e) ISGylation of FLAG-tagged MDA5 WT and K23R/K43R in HEK293T cells that were co-transfected with V5-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by FLAG-PD and IB with anti-V5 and anti-FLAG. (f) IFN-β-luciferase reporter activity in HEK293T cells that were transfected for 40 h with vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. WCLs were probed by IB with anti-FLAG and anti-Actin. (g) qRT-PCR analysis of IFNB1 and CCL5 mRNA in HEK293T cells that were transiently transfected with either vector, or increasing amounts of FLAG-tagged MDA5 WT or K23R/K43R. (h) STAT1 phosphorylation and ISG (IFIT1 and 2) protein abundance in the WCLs of HEK293T cells that were transiently transfected with vector or FLAG-tagged MDA5 WT or K23R/K43R, determined by IB with anti-pY701-STAT1, anti-STAT1, anti-IFIT1, anti-IFIT2, anti-FLAG (expression control) and anti-Actin (loading control). (i) qRT-PCR analysis of IFNB1, CCL5, OAS1, and RSAD2 mRNA in MDA5 KO SVGAs that were reconstituted with either empty vector or FLAG-tagged MDA5 WT, K23R/K43R or S88E. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f, g, and i). *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t-test). NS, not significant.