ISG15-dependent Activation Of The Sensor MDA5 Is Antagonized By The SARS-CoV-2 Papain-like Protease To Evade Host Innate Immunity

Activation of the RIG-I-like receptors, retinoic-acid inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15, the mechanistic roles of which in innate immunity still remain enigmatic. In the present study, we report that ISG15 conjugation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization 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 of SARS-CoV-2, a recently emerged coronavirus that has caused the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response and also identifies a key immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.

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Viral perturbation of host immune homeostasis is monitored by the innate immune system, which relies on receptors that sense danger- or pathogen-associated molecular patterns (PAMPs)1–3. The RIG-I-like receptors (RLRs) RIG-I and MDA5 are pivotal for virus detection by surveying the cytoplasm for viral or host-derived immunostimulatory RNAs4. The binding of RNA to the C-terminal domain (CTD) and helicase of RIG-I and MDA5 leads to their signalling-primed conformation that allows for the recruitment of several enzymes5. These enzymes modify RLRs at multiple domains and sites, and post-translational modifications (PTMs) are particularly well studied for the caspase activation and recruitment domains (CARDs), the signaling modules. Protein phosphatase 1 (PP1) / dephosphorylates the RIG-I and MDA5 CARDs6 . In the case of RIG-I, dephosphorylation promotes Lys63-linked polyubiquitination of the CARDs by TRIM25 (tripartite motif containing 25) and other E3 ligases7,8, which stabilizes the oligomeric form of RIG-I, thereby enabling mitochondrial antiviral-signalling protein (MAVS) binding. Compared with those of RIG-I, the individual steps of MDA5 activation and critical PTMs involved are less well understood. RLR activation induces the production of type I and III IFNs which, in turn, propagate antiviral signaling by upregulating ISGs9,10. Among those is ISG15, a ubiquitin-like protein that can be covalently conjugated to lysine residues of target proteins, a PTM process termed ISGylation11. Although ISG15 conjugation has been widely recognized to act antivirally12, mechanisms of host protein ISGylation that could explain the broad antiviral restriction activity of ISG15 are currently unknown. The causative agent of the ongoing COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SCoV2), belongs to the Coronaviridae family which contains several other human pathogens. Coronaviruses have an exceptional capability to suppress IFN-mediated antiviral responses, and low IFN production in SCoV2-infected patients correlated with severe disease13. Among the coronaviral IFN antagonists is the papain-like protease (PLpro) which has deubiquitinating and de-ISGylating activities14,15. In the present study, we identify an essential role for ISGylation in MDA5 activation. We further show that SCoV2 PLpro interacts with MDA5 and antagonizes ISG15-dependent MDA5 activation via active de-ISGylation, unveiling that SCoV2 has already evolved to escape immune surveillance by MDA5. 

MDA5, but not RIG-I, signaling requires ISG15. 

To identify PTMs of the MDA5 CARDs that may regulate MDA5 activation, we subjected affinity-purified MDA5–2CARD fused to glutathione-S-transferase (GST–MDA5–2CARD), or GST alone, to liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), and found that, specifically, GST–MDA5–2CARD co-purified with ISG15, which appeared as two bands that migrated more slowly (by ~15 and 30 kDa) than unmodified GST–MDA5– 2CARD (Extended Data Fig. 1a). Immunoblotting (IB) confirmed that GST–MDA5–2CARD is modified by ISG15 (Extended Data Fig. 1b). We next determined the relevance of ISG15 for MDA5-induced signaling. Whereas FLAG–MDA5 expression in wild-type (WT) mouse embryonic fibroblasts (MEFs) induced IFN- messenger RNA and protein as well as Ccl5 transcripts in a dose-dependent manner, FLAG–MDA5 expression in Isg15−/− MEFs led to ablated antiviral gene and protein expression (Fig. 1a and Extended Data Fig. 1c). Similarly, antiviral gene induction by strongly diminished in ISG15 knockout (KO) HeLa (human) cells compared with WT control cells (Fig. 1b and Extended Data Fig. 1d), ruling out a species-specific effect. In contrast, FLAG–RIG-I induced comparable amounts of secreted IFN- protein as well as Ifnb1 and Ccl5 transcripts in Isg15−/− and WT MEFs (Fig. 1a and Extended Data Fig. 1c). IFNB1 and CCL5 transcripts as well as IFN- protein production by FLAG–RIG-I were similar or slightly enhanced in ISG15 KO HeLa cells compared with WT cells (Fig. 1b and Extended Data Fig. 1d), consistent with previous reports that ISGylation negatively impacts RIG-I signalling16,17

Fig. 1|ISGylation is required for MDA5, but not RIG-I, signaling. a,b, ELISA of IFN- 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 IB with anti-ISG15, anti-FLAG, and anti-actin (loading control). c, ELISA of IFN- from supernatants of WT or Isg15−/− MEFs that were mock stimulated or transfected with EMCV RNA (0.1 or 0.4 µg ml−1 ), HMW-poly(I: C) (0.5 µg ml−1 ) or RABVLe (1 pmol ml−1 ), or infected with SeV (10 haemagglutination units (HAU) ml−1 ) for 24 h. d, RT–qPCR analysis of Ifnb1, Ccl5, and Tnf 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−1 ) or RABVLe (1 pmol ml−1 ) for 6 h, assessed by IB with anti-pSer396-IRF3 and anti-IRF3. f, ELISA of IFN- 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−1 ) or RABVLe (1 pmol ml−1 ), or infected with SeV (10 HAU ml−1 ) for 16 h. g, ELISA of IFN- from the supernatants of PBMCs that were transduced for 40 h with the indicated shRNA lentiviral particles and then infected with mutEMCV (MOI=10) or SeV (200 HAU ml−1 ) for 8 h. h, RT–qPCR analysis of IFNA2 and IL-6 mRNA in PBMCs that were transduced and infected as in g. Data represent at least two independent experiments with similar results (mean ± s.d. of n= 3 biological replicates in a–d and f, and mean of n= 2 biological replicates in g and h). *P< 0.05, **P< 0.01, ***P< 0.001 (two-tailed, unpaired Student's t-test). ND, not detected; NS, not significant.

We next tested the effect of ISG15 gene deletion on the activation of endogenous MDA5 and RIG-I by their respective ligands. IFN- production as well as IFNB1, CCL5, and TNF gene expression induced by transfection of encephalomyocarditis virus (EMCV) RNA or high-molecular-weight (HMW)-poly(I: C), both of which are predominantly sensed by MDA5, were profoundly attenuated in Isg15−/− MEF, ISG15 KO HeLa and ISG15 KO HAP-1 cells compared with their respective control cells (Fig. 1c,d and Extended Data Fig. 1e–g). Importantly, the ablation of antiviral gene induction by EMCV RNA or HMW-poly(I: C) in ISG15 KO cells was not due to abrogated MDA5 gene expression; on the contrary, MDA5 mRNA expression was enhanced in ISG15 KO cells compared with WT cells (Extended Data Fig. 1f,g). In contrast to stimulation with MDA5 agonists, stimulation of Isg15−/− MEFs and ISG15 KO HeLa cells by rabies virus leader RNA (RABVLe) transfection or Sendai virus (SeV) infection, which are RIG-I stimuli, led to IFN- production and antiviral gene expression comparable to WT cells (Fig. 1c,d and Extended Data Fig. 1e). To rule out potential clonal effects that could be associated with ISG15 gene-deleted cells, we performed transient gene-silencing experiments in primary normal human lung fibroblasts (NHLFs). ISG15 silencing, similar to MDA5 knockdown, led to an almost-complete loss of phosphorylation of IFN-regulatory factor 3 (IRF3)—a hallmark of RLR-signal activation—following stimulation with EMCV RNA but not RABVLe ished IFN- production as well as antiviral transcript expression in NHLFs transfected with EMCV RNA, but not in cells stimulated with RABVLe or SeV (Fig. 1f and Extended Data Fig. 1h). Small hairpin (sh)RNA-mediated silencing of ISG15 or MDA5 in primary human peripheral blood mononuclear cells (PBMCs) also substantially reduced antiviral protein and transcript expression after infection with a recombinant mutant EMCV (mutEMCV) deficient in MDA5 antagonism18,19, compared with infected PBMCs transduced with non-targeting control shRNA (Fig. 1g,h and Extended Data Fig. 1i). By contrast, ISG15 or MDA5 depletion did not affect cytokine responses in PBMCs after SeV infection (Fig. 1g,h and Extended Data Fig. 1i). These results show that ISG15 is essential for immune signalling by MDA5, but not RIG-I. The MDA5 CARDs are ISGylated at Lys23 and Lys43. 

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To corroborate our MS analysis that identified MDA5–2CARD ISGylation, we first tested whether endogenous MDA5 is also modified by ISG15. Endogenous MDA5 was robustly ISGylated in cells transfected with HMW-poly(I: C), or infected with dengue (DENV) or Zika (ZIKV) viruses that are sensed by MDA5 (ref. 5 ) (Fig. 2a). Notably, endogenous MDA5 was also ISGylated in uninfected cells, although at very low levels (Extended Data Fig. 2a), which is consistent with previous findings that many host proteins are also ISGylated at low levels in normal (uninfected) conditions20. In cells treated with anti-IFNAR2 to block IFNAR-signalling-mediated ISG upregulation, ISG15 or MDA5 silencing led to a comparable reduction of IFNB1 gene expression after mutEMCV infection (Extended Data Fig. 2b), indicating that ISG15-dependent MDA5 signaling occurs even in the absence of IFNAR signaling.

t MDA5Δ2CARD (containing helicase and CTD), is the primary site of MDA5 ISGylation showing two prominent bands for ISGylated 2CARD (Fig. 2b). Reconstitution of ISG15 KO HeLa cells with either WT ISG15, or an un-conjugatable ISG15 mutant in which the two glycines needed for conjugation were replaced with alanine (ISG15-AA)21, demonstrated covalent ISG15 conjugation (Fig. 2c). Mutation of individual lysine residues in GST–MDA5–2CARD to arginine revealed that single-site mutation of Lys23 and Lys43 noticeably reduced ISGylation (Extended Data Fig. 2c), whereas their combined mutation (Lys23Arg/Lys43Arg) almost abolished ISGylation (Fig. 2d). Full-length FLAG–MDA5 Lys23Arg/Lys43Arg also showed markedly diminished ISGylation (Fig. 2e and Extended Data Fig. 2d); the residual ISGylation seen in FLAG–MDA5 Lys23Arg/Lys43Arg is probably due to additional minor sites in the 2CARD and/or Δ2CARD. Of note, the Lys23Arg/Lys43Arg mutation did not affect MDA5–2CARD SUMOylation5 (Extended Data Fig. 2e). Furthermore, whereas RIG-I–2CARD was robustly ubiquitinated (which represents covalent Lys63-linked ubiquitination7 ), neither MDA5–2CARD WT nor the Lys23Arg/Lys43Arg mutant showed detectable levels of ubiquitination (Extended Data Fig. 2f). Collectively, these results indicate that the MDA5 CARDs undergo ISGylation at two major sites, Lys23 and Lys43. 

CARD ISGylation is required for MDA5 activation. 

When comparing their signal-transducing ability, MDA5–2CARD Lys23Arg and Lys43Arg single-site mutants showed partially reduced IFN- promoter activation compared with WT MDA5–2CARD, whereas the Lys23Arg/Lys43Arg mutant had a profoundly reduced signaling activity, which was almost as strong as that of the signalling-defective mutants Ser88Glu and Ser88Asp6 (Extended Data Fig. 2g). In contrast, a mutant in which Lys68, which is the lysine residue that is most proximal to Lys43 and Lys23, was substituted with arginine (Lys68Arg) showed comparable ISG15 conjugation and signaling competency to WT 2CARD (Extended Data Fig. 2c,g). MDA5–2CARD Lys23Arg/Lys43Arg, in contrast to WT MDA5–2CARD, also failed to induce IRF3 dimerization (Extended Data Fig. 2h). FLAG–MDA5 Lys23Arg, Lys43Arg or Lys23Arg/Lys43Arg also showed reduced, or almost abolished, IFN- promoter-activating abilities, compared with FLAG–MDA5 WT (Fig. 2f). MDA5 Lys23Arg/Lys43Arg showed a profound signaling defect even when expressed at high amounts, whereas WT MDA5 induced antiviral transcripts in a dose-dependent manner (Fig. 2g). In agreement, STAT1 phosphorylation, a hallmark of IFNAR signaling, as well as ISG protein expression were highly induced by MDA5 WT, but not Lys23Arg/Lys43Arg (Fig. 2h). Complementation of MDA5-gene-edited human astrocytes (SVGAs) with MDA5 Lys23Arg/Lys43Arg or Ser88Glu led to greatly diminished IFNB1, CCL5 and ISG transcripts compared with cells expressing WT MDA5 (Fig. 2i and Extended Data Fig. 2i). These results demonstrate that ISGylation at Lys23 and Lys43 is essential for MDA5-mediated cytokine responses. 

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Dephosphorylation by PP1 regulates MDA5 ISGylation.

Similar to RIG-I, MDA5 is phosphorylated within the CARDs in uninfected cells, which prevents autoactivation; dephosphorylation of RIG-I and MDA5 by PP1 / is crucial for unleashing RLRs from their signaling-repressed states6,22–24. Dephosphorylation of RIG-I allows Lys63-linked ubiquitination of the CARDs, which promotes RIG-I multimerization and signalling5. The details of how CARD dephosphorylation (at Ser88) triggers MDA5 activation have remained elusive, and therefore we tested whether dephosphorylation regulates MDA5 ISGylation. Silencing of PP1 / strongly diminished MDA5–2CARD ISGylation (Extended Data Fig. 3a). Furthermore, the phosphomimetic Ser88Glu and Ser88Asp mutants had reduced ISGylation, whereas the phospho-null Ser88Ala mutant showed stronger ISGylation than WT MDA5–2CARD (Extended Data Fig. 3b). Conversely, MDA5 WT and Lys23Arg/Lys43Arg had comparable Ser88 phosphorylation (Extended Data Fig. 3c). Together, these data suggest that MDA5 dephosphorylation at Ser88 precedes CARD ISGylation. We next made use of the measles virus V protein (MeV-V), which antagonizes MDA5 Ser88 dephosphorylation through PP1 / antagonism25. MeV-V expression enhanced the Ser88 phosphorylation (indicative of ablated dephosphorylation) of GST–MDA5–2CARD or FLAG–MDA5 in a dose-dependent manner, as previously shown25. Enhanced phosphorylation by MeV-V correlated with a decline in ISGylation (Extended Data Fig. 3d,e). In contrast to WT MeV-V, a mutant MeV-V that has abolished PP1-binding and MDA5-dephosphorylation antagonism (MeV-VΔtail)25, exhibited little effect on MDA5–2CARD ISGylation (Extended Data Fig. 3f), strengthening the inhibition of ISGylation being primarily due to PP1 inhibition, and not other antagonistic effects, by MeV-V. The V proteins from Nipah and Hendra viruses (NiV-V and HeV-V) also enhanced MDA5 Ser88 phosphorylation and, correspondingly, dampened MDA5 ISGylation (Extended Data Fig. 3g,h), suggesting that several paramyxoviral V proteins inhibit MDA5 ISGylation through manipulation of Ser88 phosphorylation, although the precise mechanisms for individual V proteins remain to be determined. Taken together, these data suggest that the MDA5 CARD ISGylation is dependent on dephosphorylation at Ser88.

ISGylation promotes higher-order MDA5 assemblies. 

RLR activation requires RNA binding, RLR oligomerization, and their translocation from the cytosol to mitochondria for interaction with MAVS5. To elucidate the mechanism by which ISGylation impacts MDA5 activity, we first examined whether ISGylation affects RNA binding. Endogenous MDA5 purified from WT or Isg15−/− MEFs interacted equally well with HMW-poly(I: C) in vitro (Extended Data Fig. 4a). MDA5 WT and Lys23Arg/Lys43Arg showed comparable binding to HMW-poly(I: C), indicating that ISGylation does not affect the RNA-binding ability of MDA5 (Extended Data Fig. 4b). When we monitored the translocation of MDA5 from the cytosol to mitochondria following EMCV RNA stimulation, we found that ISG15 silencing, but not so.C transfection, abolished MDA5 translocation (Fig. 3a). In contrast, RIG-I translocation after RABVLe transfection was efficient in both ISG15-depleted and si. C-transfected cells (Fig. 3b). These data indicated that ISGylation regulates MDA5 translocation or a step upstream of it. As the cytosol-to-mitochondria translocation of MDA5 requires interaction with 14-3-3η26, we compared 14-3-3η binding of WT and mutant MDA5. The ability of MDA5 Lys23Arg/Lys43Arg to bind 14-3-3η was similar to that of WT MDA5 or the Lys68Arg mutant (Extended Data Fig. 4c). However, whereas EMCV RNA stimulation effectively induced MDA5 oligomerization in WT MEFs, the formation of MDA5 oligomers was ablated in ISG15-deficient MEFs (Fig. 3c). ISG15 knockdown in 293T cells also abolished the oligomerization of FLAG–MDA5–2CARD (Fig. 3d). Conversely, co-expression of the ISGylation machinery components, Ube1L and UbcH8, strongly enhanced MDA5–2CARD oligomerization in si. C-transfected cells, but not in ISG15-depleted cells (Fig. 3d), indicating that ISGylation is required for MDA5 oligomer formation. In support of this concept, FLAG–MDA5 Lys23Arg/Lys43Arg showed almost abolished oligomerization, whereas WT MDA5 oligomerized efficiently (Fig. 3e). We also compared the effect of the Lys23 Arg/Lys43Arg mutation with that of oligomerization-disruptive mutations that localize either to the interface between MDA5 monomers and impede RNA-binding-mediated MDA5 filamentation (Ile841Arg/Glu842Arg and Asp848Ala/Phe849Ala)27,28, or to the CARDs (Gly74Ala/Trp75Ala) and disrupt 2CARD oligomerization27. Unlike WT MDA5, the Lys23Arg/Lys43Arg mutant, similar to MDA5 Gly74Ala/Trp75Ala, showed deficient oligomerization and, consistent with this, abolished IFN- promoter-activating ability (Fig. 3f,g). Introduction of Lys23Arg/Lys43Arg into the Ile841Arg/Glu842Arg or Asp848Ala/Phe849Ala background, either of which by itself decreased MDA5 oligomerization and signaling, also abolished MDA5 oligomer formation and IFN- induction (Fig. 3f,g). As LGP2 facilitates MDA5 nucleation on double-stranded RNA and thereby MDA5 oligomerization29,30, we compared the LGP2 binding of MDA5 WT and Lys23Arg/Lys43Arg. MDA5 Lys23Arg/Lys43Arg interacted with LGP2 as efficiently as WT MDA5 (Extended Data Fig. 4d), strengthening the proposal that CARD ISGylation promotes MDA5 oligomerization independently of RNA-binding-mediated filamentation. Collectively, these results establish that ISGylation facilitates CARD oligomerization and higher-order MDA5 assemblies.

Fig. 2|MDA5 activation requires ISGylation at Lys23 and Lys43. an Endogenous MDA5 ISGylation in NHLFs that were mock treated, transfected with HMW-poly(I: C) (0.1 µg ml−1 ) for 40 h (left) or infected with DENV or ZIKV (MOI=1 for each) for 48 h (right), determined by IP with anti-MDA5 (or an IgG isotype control) and IB with anti-ISG15. b, ISGylation of FLAG-tagged MDA5–2CARD and MDA5Δ2CARD in transiently transfected HEK293T cells that also expressed V5–ISG15, HA–Ube1L and FLAG–UbcH8, assessed by FLAG PD and IB with anti-V5 at 40 h post-transfection. 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- treatment (1,000 U ml−1 ) for 24 h, determined by IP with anti-MDA5 and IB with anti-ISG15. d, ISGylation of GST–MDA5–2CARD WT and Lys23Arg/ Lys43Arg 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. e, ISGylation of FLAG–MDA5 WT and Lys23Arg/Lys43Arg in HEK293T cells that were co-transfected with V5–ISG15, HA–Ube1L and FLAG–UbcH8, determined by FLAG PD and IB with anti-V5. f, IFN- luciferase reporter activity in HEK293T cells that were transfected for 40 h with vector, FLAG–MDA5 WT or mutants. Luciferase values are 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, RT–qPCR analysis of IFNB1 and CCL5 mRNA in HEK293T cells that were transiently transfected with either vector or increasing amounts of FLAG–MDA5 WT or Lys23Arg/Lys43Arg. h, STAT1 phosphorylation and ISG (IFIT1 and -2) protein abundance in the WCLs of HEK293T cells that were transiently transfected with vector or FLAG–MDA5 WT or Lys23Arg/Lys43Arg, determined by IB. I, RT–qPCR analysis of the indicated antiviral genes in MDA5 KO SVGAs that were transiently reconstituted with either empty vector or FLAG-tagged MDA5 WT, Lys23Arg/Lys43Arg or Ser88Glu. Data represent at least two independent experiments with similar results (mean ± s.d. of n= 3 biological replicates in f, g, and i). *P< 0.05, **P< 0.01, ***P< 0.001 (two-tailed, unpaired Student's t-test).

Fig. 3|CARD ISGylation promotes the 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−1 ) (a) or RABVLe (1 pmol ml−1 ) (b) for 16 h. IB was performed with anti-MDA5 (a), anti-RIG-I (b), anti-ISG15, and anti-actin (a and b). -Tubulin and MAVS served as purity markers for the cytosolic and mitochondrial fraction, respectively (a and b). c, Endogenous MDA5 oligomerization in WT and Isg15−/− MEFs that were transfected with EMCV RNA (0.5 µg ml−1 ) for 16 h, and 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, either with or without HA–Ube1L and FLAG–UbcH8 for 48 h, determined by NativePAGE 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 Lys23Arg/Lys43Arg 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 NativePAGE and IB with anti-MDA5. WCLs were further analyzed by SDS–PAGE and probed by IB with anti-MDA5 and anti-actin. g, IFN- 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 represent at least two independent experiments with similar results (mean ± s.d. of n= 3 biological replicates in g). ***P< 0.001 (two-tailed, unpaired Student's t-test).

ISGylation-dependent MDA5 signaling restricts virus replication. 

We next assessed whether ISGylation of MDA5 is required for its ability to restrict virus replication. FLAG–MDA5 WT, but not Lys23Arg/Lys43Arg, potently (by ~2log) inhibited EMCV replication (Fig. 4a). Similarly, MDA5 KO HEK293 cells reconstituted with WT MDA5, but not cells complemented with the Lys23Arg/ Lys43Arg mutant, effectively restricted DENV replication (Fig. 4b). We also reconstituted MDA5 KO astrocyte SVGAs, a physiologically relevant cell type for ZIKV infection, with either vector, or MDA5 WT or Lys23Arg/Lys43Arg, and then assessed ZIKV replication over a 40-h time course. ZIKV replication was attenuated by ~100-fold in cells reconstituted with WT MDA5 compared with vector-expressing cells. In contrast, cells complemented with MDA5 Lys23Arg/Lys43Arg did not restrict ZIKV, similar to cells expressing MDA5 Ser88Glu (Fig. 4c). WT MDA5, but not Lys23Arg/Lys43Arg, also restricted SCoV2 replication, although to a lesser extent than that seen for the other viruses tested (Fig. 4d).

Fig. 4|ISGylation is required for viral restriction by MDA5. EMCV titres in the supernatant of HEK293T cells that were transfected for 40 h with either vector, or FLAG–MDA5 WT or Lys23Arg/Lys43Arg, 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 transfected for 24 h with either vector, or FLAG–MDA5 WT or Lys23Arg/Lys43Arg, and then mock treated or infected with DENV (MOI=5) for 48 h, assessed by FACS using anti-flavivirus E (4G2). SSC, side scatter. c, ZIKV titers in the supernatant of MDA5 KO SVGAs that were transfected for 30 h with vector or FLAG-tagged MDA5 WT, Lys23Arg/Lys43Arg or Ser88Glu and then infected with ZIKV (MOI=0.1) for the indicated times, determined by plaque assay. p.f.u., plaque-forming units; h.p.i., hours post-infection. d, SCoV2 titers in the supernatant of HEK293T–hACE2 cells that were transfected for 24 h with either empty vector, or FLAG–MDA5 WT or Lys23Arg/Lys43Arg, 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. Sup., supernatant. 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 supernatants were UV-inactivated and transferred onto Vero 'recipient' cells. After 24 h, cells were infected with ZIKV (MOI=0.002–2) for 72 h, and ZIKV-positive cells were determined by immunostaining with anti-flavivirus E (4G2) and TrueBlue peroxidase substrate. g, RIG-I KO HEK293 'donor' cells were transfected with si. C or si.ISG15 together with either vector or FLAG–MDA5 WT or Lys23Arg/Lys43Arg, for 24 h, followed by EMCV infection (MOI=0.001) for 16 h. UV-inactivated cell supernatants were transferred onto Vero 'recipient' cells for 24 h, followed by infection with EMCV (MOI=0.001–0.1) for 40 h. EMCV-induced cytopathic effects were visualized by Coomassie Blue staining. Data represent at least two independent experiments with similar results (mean ± s.d. of n= 3 biological replicates in a–d). *P< 0.05, **P< 0.01 (two-tailed, unpaired Student's t-test).

We next determined the effect of ISG15 silencing on MDA5's ability to inhibit virus replication. Although MDA5 Lys23Arg/ Lys43Arg failed to suppress EMCV replication regardless of ISG15 silencing, WT MDA5 effectively restricted EMCV replication in si. C-transfected cells and, unexpectedly, also in ISG15-depleted cells (Extended Data Fig. 5a). In an exploration of the underlying mechanism of these unexpected results, we found that the EMCV-infected cells that expressed WT MDA5 had markedly enhanced levels of ISG protein expression when ISG15 was silenced compared with infected cells transfected with WT MDA5 and si. C (Extended Data Fig. 5b). Similarly, elevated ISG transcript and protein expression were observed in ISG15-deficient cells that were transfected with EMCV RNA or infected with mutEMCV, despite abrogation of IFN- induction (Extended Data Fig. 5c,d). In contrast, MDA5 knockdown abrogated both IFN- and ISG protein expression, as expected (Extended Data Fig. 5d). We noticed that the protein abundance of USP18, a deubiquitinating enzyme that negatively regulates IFNAR signaling31, was greatly diminished in ISG15-depleted cells following EMCV infection compared with infected cells that were transfected with si. C or MDA5-specific siRNA (Extended Data Fig. 5b,d), which is consistent with the reported role of ISG15 in preventing USP18 degradation32. Together, these data suggest that in experimental settings of ISG15-gene targeting (that is, silencing or KO) the antiviral effect of MDA5 ISGylation is masked by aberrant ISG upregulation due to the ablation of ISG15's inhibitory effect on IFNAR signaling.

Fig. 5|SCoV2 PLpro binds to and de-ISGylates MDA5–2CARD. a Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (Protein Data Bank, accession no. 6YVA). Key residues that mediate site 1 interaction (Asn156 and Arg166/Glu167) or site 2 interaction (Phe69) in PLpro, as well as its catalytically active site (Cys111), 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-tagged SCoV2– PLpro or FLAG-tagged MeV-V (positive 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-tagged SCoV2 PLpro WT or Cys111Ala for 24 h, assessed by NativePAGE and IB with anti-FLAG. WCLs were further analysed 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 proteins, determined by GST PD and IB with anti-FLAG, anti-V5 and anti-GST. Data represent at least two independent experiments with similar results.

We next employed a virus protection assay that experimentally decouples MDA5 signaling in virus-infected cells from downstream IFNAR signaling in the same cells (Fig. 4e). Supernatants from mutEMCV-infected 'donor' cells that were either si. C transfected, or depleted of either ISG15 or MDA5, were ultraviolet (UV) inactivated and then transferred on to uninfected 'recipient' cells. 'Primed' recipient cells were then infected with ZIKV to directly monitor the antiviral effect of MDA5-mediated IFN production by donor cells. Whereas the supernatants from si. C-transfected 'donor' cells potently inhibited ZIKV replication, and the supernatants from ISG15 or MDA5 knockdown cells minimally restricted ZIKV infection (Fig. 4f). Similarly, the culture supernatants from EMCV-infected donor cells transfected with WT MDA5 together with si. C led to greater protection of recipient cells from the viral challenge than that from cells expressing WT MDA5 and depleted of ISG15 (Fig. 4g). Collectively, these data demonstrate that ISGylation is important for MDA5-mediated restriction of a range of RNA viruses.

Fig. 6|SCoV2 PLpro inhibits ISG15-mediated MDA5 signaling via its de-ISGylase activity. a, RT–qPCR 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−1 ) 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-Nsp3 and anti-MDA5. WCLs were probed by IB with anti-Nsp3 and anti-actin. c, Endogenous MDA5 ISGylation in A549–hACE2 cells that were mock infected or infected with SCoV2 (MOI=0.5) for 40 h in the presence of PLpro inhibitor (GRL-0617; 50 µM) or vehicle control (dimethylsulfoxide), determined by IP with anti-MDA5 (or an IgG isotype control), followed by IB with anti-ISG15 and anti-MDA5. Protein abundance of IFIT1, RSAD2, ISG15, and actin in the WCLs was probed by IB. Efficient virus replication was verified by IB with anti-Nsp3 and anti-Spike (S). d, RT–qPCR analysis of IFNB1, CCL5, and IFIT1 transcripts, 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-tagged SCoV2 PLpro WT, Cys111Ala or Arg166Ser/Glu167Arg, 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 represent at least two independent experiments with similar results (mean ± s.d. of n= 3 biological replicates in a, d, and e). *P< 0.05, **P< 0.01, ***P< 0.001 (two-tailed, unpaired Student's t-test).

SCoV2 PLpro targets MDA5 for de-ISGylation.

Coronaviruses such as SARS-CoV (SCoV), MERS–CoV and the recently emerged SCoV2 encode a PLpro that mediates viral polyprotein cleavage33. In addition, PLpro has deubiquitinating and de-ISGylating activities. SCoV2 PLpro was recently shown to modulate antiviral responses primarily via its de-ISGylase activity15. As MDA5 is known to be a major sensor for detecting coronaviruses34,35, and because our data showed that ISGylation is required for MDA5-mediated virus restriction, we examined whether SCoV2 PLpro enzymatically removes MDA5 ISGylation to antagonize innate immunity. SCoV2 PLpro WT, but not its catalytically inactive mutant (PLpro Cys111Ala)15, abolished the ISGylation of GST–MDA5–2CARD and FLAG–MDA5 (Fig. 5a,b and Extended Data Fig. 6a). The PLpro Asn156Glu and Arg166Ser/Glu167Arg mutants, which are marginally and severely impaired in ISG15 binding at the 'site 1' interface, respectively14,36, did slightly, or not, affect ISGylation. In contrast, PLpro Phe69Ala, in which the 'site 2' interface that preferentially determines binding to ubiquitin, but not ISG15, is disrupted14,36, diminished MDA5 ISGylation as potently as WT PLpro (Fig. 5a,b and Extended Data Fig. 6a). SCoV2 PLpro did not, however, suppress RIG-I–2CARD ubiquitination (Extended Data Fig. 6b). We found that PLpro interacted specifically with MDA5, but not RIG-I, as did MeV-V which binds MDA5 and served as a control37 (Fig. 5c). Low amounts of PLpro inhibited signaling by MDA5, but not RIG-I, whereas higher amounts of PLpro suppressed antiviral signaling by both RLRs (Extended Data Fig. 6c). This strengthens MDA5 being a direct target of PLpro. De-ISGylation of IRF3 probably accounts for the inhibitory effect that higher doses of PLpro have on RLR signalling15,38. When examining the effect of PLpro on MDA5–2CARD oligomerization, PLpro WT but not Cys111Ala efficiently blocked MDA5–2CARD oligomerization (Fig. 5d), indicating that SCoV2 PLpro inhibits the ISGylation-dependent MDA5 oligomer formation via its enzymatic activity. The PLpro enzymes of the related -coronaviruses, SCoV, MERS–CoV and murine hepatitis virus (MHV), as well as of the -coronavirus HCoV-NL63 (NL63) also bound to and efficiently reduced MDA5–2CARD ISGylation (Fig. 5e), suggesting that MDA5 antagonism by PLpro may be widely conserved among coronaviruses. 

cistanche plant-increasing immune system

SCoV2 PLpro antagonizes ISG15-dependent MDA5 signaling. 

We next determined the relevance of ISG15-dependent MDA5 signaling for antiviral cytokine induction elicited by SCoV2. As SCoV2 infection is known to minimally induce type I IFNs due to effective viral antagonisms39, we isolated total RNA from SCoV2-infected cells and then re-transfected it into cells to stimulate innate immune signaling. SCoV2 RNA, but not RNA from mock-treated cells, robustly induced IFN transcripts; however, this induction was markedly diminished when ISG15 or MDA5 was silenced (Fig. 6a). RIG-I knockdown did not adversely affect the antiviral gene expression elicited by SCoV2 RNA, indicating that SCoV2 RNA–PAMPs are primarily sensed by the ISG15–MDA5 axis (Fig. 6a).

We found that SCoV2 non-structural protein 3 (Nsp3), within which PLpro lies, readily interacted with endogenous MDA5 during authentic SCoV2 infection (Fig. 6b). Endogenous MDA5 ISGylation was undetectable in SCoV2-infected cells, although the virus triggered ISG15 expression; however, in infected cells treated with a specific PLpro inhibitor15, MDA5 ISGylation and downstream ISG induction were strongly enhanced (Fig. 6c), supporting the proposal that PLpro effectively suppresses MDA5 ISGylation and signaling during live SCoV2 infection. We next examined the effect of WT and mutant PLpro on the activation of endogenous MDA5 during mutEMCV infection. Consistent with their effect on MDA5 ISGylation (Fig. 5b and Extended Data Fig. 6a), SCoV2 PLpro WT and Phe69Ala prevented antiviral transcript induction, whereas MDA5 Arg166Ser/ Glu167Arg, similar to the Cys111Ala mutant, did not affect antiviral gene expression (Fig. 6d). In agreement with this, mutEMCV replication was enhanced in cells expressing PLpro WT or Phe69Ala, but not in cells expressing PLpro Cys111Ala or Arg166Ser/Glu167Arg (Fig. 6d). Likewise, WT PLpro, but not the Arg166Ser/Glu167Arg or Cys111Ala mutant, blocked EMCV restriction by FLAG–MDA5 (Fig. 6e); the effect on virus replication correlated with induced ISG proteins (Fig. 6f). Collectively, this establishes SCoV2 PLpro as an IFN antagonist that actively de-ISGylates MDA5.

ISG15 conjugation is known to confer antiviral activity to a multitude of viruses; however, only a few genuine substrates have been identified12. On the other hand, ISG15 in its unconjugated form acts provirally by fortifying USP18-mediated IFNAR-signal inhibition31,32,40. The present study identifies a key role for ISGylation in MDA5-mediated IFN induction. Our work also stresses the importance of experimental design in which decoupling the role of ISG15 in MDA5 activation from that in dampening IFNAR signaling is essential to reveal ISG15's potent antiviral activity. In an infected organism, it is probably the sum of multiple ISGylation events (affecting both host and viral proteins) that determines the outcome of infection and pathogenesis, which may be context-dependent12.

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Our findings indicate that ISGylation of MDA5 acts analogously to the Lys63-linked ubiquitination of RIG-I5: both PTMs (1) are regulated by PP1-induced dephosphorylation and (2) promote CARD oligomerization and RLR higher-order assemblies. However, whereas ubiquitin is abundant in both uninfected and infected cells, ISG15 expression is strongly increased by IFN stimulation. Nevertheless, even at basal levels, ISG15 is conjugated to many host proteins20, including MDA5 as our work showed, which may be sufficient for initial MDA5 activation. During viral infections that are sensed by multiple PRRs, MDA5 ISGylation may be a 'priming' mechanism whereby ISG15 upregulation by an immediate innate sensor (for example, RIG-I)41 primes MDA5 to enter a 'kick-start' mode. As ISG15 negatively regulates RIG-I16,17, ISGylation may trigger 'sensor switching' where MDA5 activation is promoted when ISG15 levels increase, while RIG-I activity is being dampened. We identified that SCoV2 PLpro antagonizes MDA5 ISGylation via its enzymatic activity after binding to the sensor; this strategy is probably conserved among coronaviruses, which warrants further investigation. Cryo-electron microscopy analyses revealed that coronaviral Nsp3 is part of a pore complex that spans endoplasmic reticulum-derived double-membrane vesicles and exports newly synthesized viral RNA42. Thus, MDA5 may position itself in close proximity to the site of viral RNA export to facilitate PAMP detection; however, the PLpro domain of Nsp3 (which is on the cytoplasmic side) blocks MDA5 signaling through direct de-ISGylation. Some viruses may also inhibit MDA5 ISGylation through dysregulation of MDA5 phosphorylation, as shown for MeV-V. In summary, our study uncovers a prominent role for ISGylation in activating MDA5-mediated immunity as well as its inhibition by SCoV2, unveiling a potential molecular target for the design of therapeutics against COVID-19.

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