ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

Abstract

Activation of the RIG-I-like receptors, RIG-I and MDA5, establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15 whose mechanistic roles in innate immunity still remain enigmatic. Here we report that ISGylation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISG15 conjugation to the caspase activation and recruitment domains of MDA5 promotes the formation of higher-order assemblies of MDA5 and thereby triggers activation of innate immunity against a range of viruses including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease (PLpro) of SARS-CoV-2, a recently emerged coronavirus that causes the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a novel immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.

Figure 1.. ISGylation is required for MDA5-, but not RIG-I, signaling.

(a, b) ELISA of IFN-β from supernatants of MEFs (WT or Isg15^−/−) (a) and HeLa cells (WT or ISG15 KO) (b) transiently transfected with increasing amounts of FLAG-tagged MDA5 or RIG-I for 40 h. Whole cell lysates (WCLs) were probed by immunoblotting (IB) with anti-ISG15, anti-FLAG, and anti-Actin (loading control). (c) ELISA of IFN-β from supernatants of WT or Isg15^−/− MEFs that were mock-stimulated or transfected with EMCV-RNA (0.1 or 0.4 μg/mL), HMW-poly (I:C) (0.5 μg/mL), or RABV_Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 24 h. (d) Quantitative RT-PCR (qRT-PCR) analysis of IFNB1 and CCL5 mRNA in WT and Isg15^−/− MEFs stimulated as in (c). (e) IRF3 phosphorylation in the WCLs of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV_Le (1 pmol/mL) for 6 h, assessed by IB with anti-pS396-IRF3 and anti-IRF3. (f) ELISA of IFN-β from supernatants of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV_Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 16 h. (g) ELISA of IFN-β from the supernatants of PBMCs that were transduced for 40 h with the indicated shRNAs and then infected with mutEMCV (MOI 10) or SeV (200 HAU/mL) for 8 h. (h) qRT-PCR analysis of IFNA2 and IL-6 mRNA in PBMCs that were transduced and infected as in (g). Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c, d, f and mean of n = 2 biological replicates in g and h). *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t-test). ND, not detected; NS, not significant.

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

(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.

Figure 3.. CARD ISGylation is essential for formation of higher-order MDA5 assemblies.

(a,b) Cytosol-mitochondria fractionation of WCLs from NHLFs that were transfected for 30 h with non-targeting control siRNA (si.C) or ISG15-specific siRNA (si.ISG15) and then mock-treated or transfected with EMCV-RNA (0.4 μg/mL) (a) or RABV_Le (1 pmol/mL) (b) for 16 h. IB was performed with anti-MDA5 (a), anti-RIG-I (b), anti-ISG15 and anti-Actin (a, b). α-Tubulin and MAVS served as purity markers for the cytosolic and mitochondrial fraction, respectively (a, b). (c) Endogenous MDA5 oligomerization in WT and Isg15^−/− MEFs that were transfected with EMCV-RNA (0.5 μg/mL) for 16 h, assessed by SDD-AGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (d) Oligomerization of FLAG-MDA5–2CARD in HEK293T cells that were transfected with the indicated siRNAs together with or without HA-Ube1L and FLAG-UbcH8 for 48 h, determined by native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-HA, anti-ISG15, and anti-Actin. (e) Oligomerization of FLAG-MDA5 WT and K23R/K43R in transiently transfected MDA5 KO HEK293 cells, assessed by SDD-AGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and IB with anti-FLAG and anti-Actin. (f) Oligomerization of FLAG-tagged MDA5 WT and mutants in transiently transfected MDA5 KO HEK293 cells, assessed by native PAGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (g) IFN-β-luciferase reporter activity in MDA5 KO HEK293 cells that were transfected for 24 h with either empty 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. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f). ***p < 0.001 (unpaired Student’s t-test).

Figure 4.. ISGylation is required for viral restriction by MDA5.

(a) EMCV titers in the supernatant of HEK293T cells that were transiently transfected for 40 h with either empty vector, or FLAG-tagged MDA5 WT or K23/K43R and then infected with EMCV (MOI 0.001) for 24 h, determined by TCID50 assay. (b) Percentage of DENV-infected MDA5 KO HEK293 cells that were transiently transfected for 24 h with either empty vector or FLAG-tagged MDA5 WT or K23R/K43R and then mock-treated or infected with DENV (MOI 5) for 48 h, assessed by FACS using an anti-flavivirus E (4G2) antibody. SSC, side scatter. (c) ZIKV titers in the supernatant of MDA5 KO SVGAs that were transiently transfected for 30 h with either empty vector, or FLAG-tagged MDA5 WT, K23R/K43R, or S88E and then infected with ZIKV (MOI 0.1) for the indicated times, determined by plaque assay. (d) SCoV2 titers in the supernatant of HEK293T-hACE2 cells that were transiently transfected for 24 h with either empty vector, or FLAG-tagged MDA5 WT or K23/K43R and then infected with SCoV2 (MOI 0.5) for 24 h, determined by plaque assay. (e) Schematic of the experimental approach to decouple the role of ISG15 in MDA5-mediated IFN induction from its role in dampening IFNAR signaling. (f) NHLF ‘donor’ cells were transfected for 40 h with the indicated siRNAs and then infected with mutEMCV (MOI 0.1) for 16 h. Cell culture supernatants were UV-inactivated and transferred onto Vero ‘recipient’ cells for 24 h, followed by infection of cells with ZIKV (MOI 0.002 to 2) for 72 h. ZIKV-positive cells were determined by immunostaining with anti-flavivirus E (4G2) antibody and visualized using the KPL TrueBlue peroxidase substrate. (g) RIG-I KO HEK293 ‘donor’ cells were transfected for 24 h with si.C or si.ISG15 and subsequently transfected with either empty vector or FLAG-tagged MDA5 WT or K23R/K43R for 24 h, followed by EMCV infection (MOI 0.001) for 16 h. UV-inactivated culture supernatants were transferred onto Vero ‘recipient’ cells for 24 h, followed by infection with EMCV (MOI 0.001 to 0.1) for 40 h. EMCV-induced cytopathic effects were visualized by Coomassie blue staining. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c). **p < 0.01 (unpaired Student’s t-test).

Figure 5.. SCoV2 PLpro binds to and de-ISGylates MDA5–2CARD.

(a) Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (PDB: 6YVA). Key residues that mediate ‘site 1’ interaction (N156 and R166/E167) or ‘site 2’ interaction (F69) in PLpro, as well as its catalytically-active site (C111), are indicated. (b) ISGylation of GST-MDA5–2CARD in HEK293T cells that were co-transfected for 20 h with vector or V5-tagged SCoV2 PLpro WT or mutants, along with FLAG-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by GST-PD and IB with anti-FLAG and anti-GST. WCLs were probed by IB with anti-V5, anti-HA, anti-FLAG, and anti-Actin. (c) Binding of HA-tagged MDA5 or RIG-I to V5-SCoV2-PLpro or FLAG-MeV-V (control) in transiently transfected HEK293T cells, determined by HA-PD and IB with anti-V5 or anti-FLAG, and anti-HA. WCLs were probed by IB with anti-V5 and anti-FLAG. (d) Oligomerization of FLAG-MDA5–2CARD in HEK293T cells that were co-transfected with vector, or V5-SCoV2 PLpro WT or C111A for 24 h, assessed by Native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-V5 and anti-Actin. (e) ISGylation of GST-MDA5–2CARD in HEK293T cells that also expressed FLAG-ISG15, HA-Ube1L and FLAG-UbcH8, and were co-transfected for 40 h with vector or the indicated V5-tagged coronaviral PLpro, determined by GST-PD and IB with anti-FLAG, anti-V5, and anti-GST. Data are representative of at least two independent experiments.

Figure 6.. SCoV2 PLpro inhibits ISG15-mediated MDA5 signaling via its deISGylase activity.

(a) qRT-PCR analysis of IFNB1, IFNL1, ISG15, MDA5, and RIG-I transcripts in NHLFs that were transfected with the indicated siRNAs for 40 h and then transfected with mock-RNA or SCoV2-RNA (0.4 μg/mL) for 24 h. (b) Binding of SCoV2 Nsp3 to endogenous MDA5 in A549-hACE2 cells that were infected with SCoV2 (MOI 0.5) for 24 h, determined by IP with anti-MDA5 (or an IgG isotype control) followed by IB with anti-PLpro and anti-MDA5. WCLs were probed by IB with anti-PLpro (Nsp3) and anti-Actin. (c) Endogenous MDA5 ISGylation in A549-hACE2 cells that were mock-infected, or infected with SCoV2 or DENV (MOI 0.1 for each) for 48 h, determined by immunoprecipitation (IP) with anti-MDA5 followed by IB with anti-ISG15 and anti-MDA5. Protein abundance of IFIT1, RSAD2, IFITM3, ISG15 and actin in the WCLs were probed by IB. Efficient virus replication was verified by immunoblotting WCLs with anti-PLpro (Nsp3) or anti-NS3 (DENV). (d) qRT-PCR analysis of IFNB1, CCL5, IFIT1 transcript, and EMCV genomic RNA (gRNA) in HeLa cells that were transiently transfected for 24 h with vector, or V5-SCoV2 PLpro WT or mutants and then infected with mutEMCV (MOI 0.5) for 12 h. (e) EMCV titers in the supernatant of RIG-I KO HEK293 cells that were transiently transfected for 24 h with vector or FLAG-MDA5 along with V5-SCoV2 PLpro WT, C111A, or R166S/E167R and then infected with EMCV (MOI 0.001) for 16 h, determined by plaque assay. (f) Protein abundance of the indicated ISGs in the WCLs from the experiment in (e), determined by IB with the indicated antibodies. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, d, e). *p < 0.05, ***p < 0.001 (unpaired Student’s t-test).