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ISG15-Dependent Activation of the RNA Sensor MDA5 and Its Antagonism By bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by 2 the SARS-CoV-2 papain-like protease 3 4 GuanQun Liu1,2†, Jung-Hyun Lee1,2†, Zachary M. Parker2, Dhiraj Acharya1,2, Jessica J. Chiang3, 5 Michiel van Gent1,2, William Riedl1,2, Meredith E. Davis-Gardner3, Effi Wies3, Cindy Chiang1,2, 6 and Michaela U. Gack1,2* 7 8 1Florida Research and Innovation Center, Cleveland Clinic, FL 34987, USA. 9 2Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA. 10 3Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA. 11 †These authors contributed equally to this work. 12 13 *Correspondence should be addressed to M.U.G. ([email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 14 ABSTRACT 15 Activation of the RIG-I-like receptors, RIG-I and MDA5, establishes an antiviral state by 16 upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15 whose mechanistic 17 roles in innate immunity still remain enigmatic. Here we report that ISGylation is essential for 18 antiviral IFN responses mediated by the viral RNA sensor MDA5. ISG15 conjugation to the 19 caspase activation and recruitment domains of MDA5 promotes the formation of higher-order 20 assemblies of MDA5 and thereby triggers activation of innate immunity against a range of viruses 21 including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of 22 MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease (PLpro) 23 of SARS-CoV-2, a recently emerged coronavirus that causes the COVID-19 pandemic. Our work 24 demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies 25 a novel immune evasion mechanism of SARS-CoV-2, which may be targeted for the development 26 of new antivirals and vaccines to combat COVID-19. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 INTRODUCTION 28 Viral perturbation of host immune homeostasis is monitored by the innate immune system, 29 which relies on receptors that sense pathogen- or danger-associated molecular patterns1, 2, 3. The 30 RIG-I-like receptors (RLRs), RIG-I and MDA5 are pivotal for virus detection by surveying the 31 cytoplasm for viral or host-derived immunostimulatory RNAs that harbor dsRNA structures and, 32 in the case of RIG-I agonists, also a 5'-di- or tri-phosphate moiety4. Binding of RNA to the C- 33 terminal domain (CTD) and helicase of RIG-I and MDA5 leads to their transition from an inactive 34 state to a signaling-primed conformation that allows for the recruitment of several enzymes5. These 35 enzymes modify RLRs at multiple domains and sites, and posttranslational modifications (PTMs) 36 are particularly well studied for the N-terminal caspase activation and recruitment domains 37 (CARDs), the signaling modules. PP1α/γ dephosphorylate specific CARD residues in RIG-I and 38 MDA56, which triggers further activation steps. In the case of RIG-I, dephosphorylation promotes 39 K63-linked polyubiquitination of the CARDs by TRIM25 and other E3 ligases7, 8, which nucleates 40 and stabilizes the oligomeric form of RIG-I, thereby enabling MAVS binding at mitochondria. 41 Compared to those of RIG-I, the individual steps of MDA5 activation and critical PTMs involved 42 are less well understood. 43 RLR activation induces the production of type I and III interferons (IFNs) which, in turn, 44 propagate antiviral signaling by upregulating IFN-stimulated genes (ISGs)9, 10. Among the 45 profoundly upregulated ISGs is ISG15, a ubiquitin-like protein. Similar to ubiquitin, ISG15 can 46 be covalently conjugated to lysine (K) residues of target proteins, a PTM process termed 47 ISGylation11. ISGylation is catalyzed by a chain of enzymatic reactions analogous to ubiquitination, 48 involving an E1 activating enzyme (Ube1L), an E2 conjugating enzyme (UbcH8), and a handful 49 of E3 ligases (for example, HERC5). Inversely, de-ISGylation is mediated by the cellular 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 isopeptidase USP1811, and certain viruses also encode proteases that harbor de-ISGylase 51 activities12. Besides covalent conjugation, ISG15 – like ubiquitin – can noncovalently bind to 52 substrate proteins. Whereas ISG15 conjugation has been widely recognized to act antivirally13, 53 unconjugated ISG15 serves a proviral role by promoting USP18-mediated suppression of type I 54 IFN receptor (IFNAR) signaling14, 15, 16; this latter function of ISG15 is responsible for over- 55 amplified ISG induction and fortified viral resistance in humans with inherited ISG15 deficiency. 56 In contrast to ISG15’s role in dampening IFNAR signaling, the precise mechanism(s) of how 57 ISGylation enhances immune responses to a wide range of viral pathogens are less well understood. 58 Along these lines, although a broad repertoire of viral and cellular proteins has been shown to be 59 targeted for ISGylation13 (of note, this usually represents co-translational modification of the 60 nascent protein pool17), mechanisms of host protein ISGylation that could explain the broad 61 antiviral restriction activity of ISG15 are currently unknown. 62 The causative agent of the ongoing COVID-19 pandemic, severe acute respiratory 63 syndrome coronavirus 2 (SCoV2), belongs to the Coronaviridae family that contains several other 64 human pathogens. Coronaviruses have an exceptional capability to suppress IFN-mediated 65 antiviral responses, and low production of type I IFNs in SCoV-2-infected patients correlated with 66 more severe disease outcome18. Among the coronaviral IFN antagonists is the papain-like protease 67 (PLpro), which has deubiquitinating and de-ISGylating activities19, 20; however, the cellular 68 substrates of the SCoV2 PLpro remain largely elusive. 69 Here we identify an essential role for ISGylation in MDA5 activation. We further show 70 that SCoV2 PLpro interacts with MDA5 and antagonizes ISG15-dependent MDA5 activation via 71 its de-ISGylase activity, unveiling that SCoV2 has already evolved to escape immune surveillance 72 by MDA5. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 73 RESULTS 74 MDA5, but not RIG-I, signaling requires ISG15 75 To identify PTMs of the CARDs of MDA5 that may regulate MDA5 activation, we 76 subjected affinity-purified MDA5-2CARD fused to glutathione-S-transferase (GST-MDA5- 77 2CARD), or GST alone, to liquid chromatography coupled with tandem mass spectrometry (LC- 78 MS/MS) and found that specifically GST-MDA5-2CARD co-purified with ISG15, which 79 appeared as two bands that migrated more slowly (by ~15 and 30 kDa) than unmodified GST- 80 MDA5-2CARD (Extended Data Fig. 1a). Immunoblot (IB) analysis confirmed that GST-MDA5- 81 2CARD is modified by ISG15 (Extended Data Fig. 1b). Since the CARDs are the signaling 82 module of MDA5, we next determined the functional relevance of ISG15 for MDA5-induced 83 signaling. While ectopic expression of FLAG-MDA5 in wild-type (WT) mouse embryonic 84 fibroblasts (MEFs) induced IFN-β mRNA and protein as well as Ccl5 transcripts in a dose- 85 dependent manner, FLAG-MDA5 expression in Isg15-/- MEFs led to ablated antiviral gene and 86 protein expression (Fig. 1a and Extended Data Fig. 1c). Similarly, antiviral gene expression 87 induced by FLAG-MDA5 was strongly diminished in ISG15 KO HeLa (human) cells compared 88 to WT control cells (Fig. 1b and Extended Data Fig. 1d), ruling out a species-specific effect. In 89 contrast to FLAG-MDA5, ectopically expressed FLAG-RIG-I induced comparable amounts of 90 secreted IFN-β protein as well as Ifnb1 and Ccl5 transcripts in Isg15-/- and WT MEFs (Fig. 1a 91 and Extended Data Fig. 1c). IFNB1 transcripts and IFN-β protein production triggered by FLAG- 92 RIG-I were slightly enhanced in ISG15 KO HeLa cells as compared to WT control cells (Fig. 1b 93 and Extended Data Fig. 1d), consistent with previous reports that ISGylation negatively impacts 21, 22 94 RIG-I signaling . These results suggest that ISG15 is required for MDA5, but not RIG-I, 95 mediated signal transduction. 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356048; this version posted October 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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