This is an unedited manuscript that has been accepted for publication. Nature Research are providing this early version of the manuscript as a service to our authors and readers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity


The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3,4,5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

Data availability

The atomic coordinates of PLpro–mouse ISG15 have been deposited in the PDB with accession code 6YVA. The mass spectrometry data have been deposited to the ProteomeXchange Consortium74 via the PRIDE partner repository75 with the data set identifier PXD018983. The papain-like protease domain sequence was obtained from the SARS-CoV-2 complete genome (NCBI nucleotide, severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome; NC_045512). Protein sequence for SCoV2-PLpro Ubl domain (amino acids, 746–1060) of Nsp3 protein from SARS-CoV-2 (Nsp3; YP_009725299.1). Full gel images are shown in Supplementary Fig. 1. Any other relevant data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.


  1. 1.

    Harcourt, B. H. et al. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity. J. Virol. 78, 13600–13612 (2004).

    Google Scholar 

  2. 2.

    Lim, K. P., Ng, L. F. P. & Liu, D. X. Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products. J. Virol. 74, 1674–1685 (2000).

    Google Scholar 

  3. 3.

    Frieman, M., Ratia, K., Johnston, R. E., Mesecar, A. D. & Baric, R. S. Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-κB signaling. J. Virol. 83, 6689–6705 (2009).

    Google Scholar 

  4. 4.

    Devaraj, S. G. et al. Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus. J. Biol. Chem. 282, 32208–32221 (2007).

    Google Scholar 

  5. 5.

    Bailey-Elkin, B. A. et al. Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression. J. Biol. Chem. 289, 34667–34682 (2014).

    Google Scholar 

  6. 6.

    Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020).

    Google Scholar 

  7. 7.

    Lu, R. et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565–574 (2020).

    Google Scholar 

  8. 8.

    Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).

    Google Scholar 

  9. 9.

    Sommer, S., Weikart, N. D., Linne, U. & Mootz, H. D. Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition. Bioorg. Med. Chem. 21, 2511–2517 (2013).

    Google Scholar 

  10. 10.

    Ekkebus, R. et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135, 2867–2870 (2013).

    Google Scholar 

  11. 11.

    Flierman, D. et al. Non-hydrolyzable diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by S2 pockets. Cell Chem. Biol. 23, 472–482 (2016).

    Google Scholar 

  12. 12.

    Basters, A. et al. Structural basis of the specificity of USP18 toward ISG15. Nat. Struct. Mol. Biol. 24, 270–278 (2017).

    Google Scholar 

  13. 13.

    Geurink, P. P. et al. Profiling DUBs and Ubl-specific proteases with activity-based probes. Methods Enzymol. 618, 357–387 (2019).

    Google Scholar 

  14. 14.

    Freitas, B. T. et al. Characterization and noncovalent inhibition of the deubiquitinase and deISGylase activity of SARS-CoV-2 papain-like protease. ACS Infect. Dis. 6, 2009–2109 (2020).

    Google Scholar 

  15. 15.

    Basters, A. et al. Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15. FEBS J. 281, 1918–1928 (2014).

    Google Scholar 

  16. 16.

    Clasman, J. R., Everett, R. K., Srinivasan, K. & Mesecar, A. D. Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. Antiviral Res. 174, 104661 (2020).

    Google Scholar 

  17. 17.

    Daczkowski, C. M. et al. Structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species. J. Mol. Biol. 429, 1661–1683 (2017).

    Google Scholar 

  18. 18.

    Ratia, K. et al. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proc. Natl Acad. Sci. USA 105, 16119–16124 (2008).

    Google Scholar 

  19. 19.

    Báez-Santos, Y. M. et al. X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases. J. Med. Chem. 57, 2393–2412 (2014).

    Google Scholar 

  20. 20.

    Ghosh, A. K. et al. Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation. J. Med. Chem. 53, 4968–4979 (2010).

    Google Scholar 

  21. 21.

    Ghosh, A. K. et al. Structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease. J. Med. Chem. 52, 5228–5240 (2009).

    Google Scholar 

  22. 22.

    Kilianski, A. & Baker, S. C. Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral Res. 101, 105–112 (2014).

    Google Scholar 

  23. 23.

    Gordon, D. E. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459–468 (2020).

    Google Scholar 

  24. 24.

    Ferguson, B. J., Mansur, D. S., Peters, N. E., Ren, H. & Smith, G. L. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. eLife 1, e00047 (2012).

    Google Scholar 

  25. 25.

    Bojkova, D. et al. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets. Nature 583, 469–472 (2020).

    Google Scholar 

  26. 26.

    Ouellet, M. et al. Galectin-1 acts as a soluble host factor that promotes HIV-1 infectivity through stabilization of virus attachment to host cells. J. Immunol. 174, 4120–4126 (2005).

    Google Scholar 

  27. 27.

    Schick, C. et al. Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis. Biochemistry 37, 5258–5266 (1998).

    Google Scholar 

  28. 28.

    Takeda, A., Yamamoto, T., Nakamura, Y., Takahashi, T. & Hibino, T. Squamous cell carcinoma antigen is a potent inhibitor of cysteine proteinase cathepsin L. FEBS Lett. 359, 78–80 (1995).

    Google Scholar 

  29. 29.

    Stetson, D. B. & Medzhitov, R. Type I interferons in host defense. Immunity 25, 373–381 (2006).

    Google Scholar 

  30. 30.

    Liu, T., Zhang, L., Joo, D. & Sun, S.-C. NF-κB signaling in inflammation. Signal Transduct. Target. Ther. 2, 17023 (2017).

    Google Scholar 

  31. 31.

    Sheikh, F., Dickensheets, H., Gamero, A. M., Vogel, S. N. & Donnelly, R. P. An essential role for IFN-β in the induction of IFN-stimulated gene expression by LPS in macrophages. J. Leukoc. Biol. 96, 591–600 (2014).

    Google Scholar 

  32. 32.

    Shi, H.-X. et al. Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification. Mol. Cell. Biol. 30, 2424–2436 (2010).

    Google Scholar 

  33. 33.

    Blanco-Melo, D. et al. Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 181, 1036–1045 (2020).

    Google Scholar 

  34. 34.

    Niemeyer, D. et al. The papain-like protease determines a virulence trait that varies among members of the SARS-coronavirus species. PLoS Pathog. 14, e1007296 (2018).

    Google Scholar 

  35. 35.

    Anson, B. J. et al. Broad-spectrum inhibition of coronavirus main and papain-like 2 proteases by HCV drugs. Preprint at (2020).

  36. 36.

    Shanker, A. K., Bhanu, D., Alluri, A. & Gupta, S. Whole-genome sequence analysis and homology modelling of the main protease and non-structural protein 3 of SARS-CoV-2 reveal an aza-peptide and a lead inhibitor with possible antiviral properties. New J. Chem. 44, 9202–9212 (2020).

    Google Scholar 

  37. 37.

    Rut, W. et al. Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design. Preprint at bioRxiv (2020).

  38. 38.

    Zhang, L. et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science 368, 409–412 (2020).

    Google Scholar 

  39. 39.

    Jin, Z. et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors. Nature 582, 289–293 (2020).

    Google Scholar 

  40. 40.

    Dai, W. et al. Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science 368, 1331–1335 (2020).

    Google Scholar 

  41. 41.

    Lo, H. S. et al. Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir. Prepint at bioRxiv (2020).

  42. 42.

    Daczkowski, C. M., Goodwin, O. Y., Dzimianski, J. V., Farhat, J. J. & Pegan, S. D. Structurally guided removal of DeISGylase biochemical activity from papain-like protease originating from Middle East respiratory syndrome coronavirus. J. Virol. 91, e01067-17 (2017).

    Google Scholar 

  43. 43.

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Google Scholar 

  44. 44.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    Google Scholar 

  45. 45.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Google Scholar 

  46. 46.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Google Scholar 

  47. 47.

    Baek, K. et al. NEDD8 nucleates a multivalent cullin-RING–UBE2D ubiquitin ligation assembly. Nature 578, 461–466 (2020).

    Google Scholar 

  48. 48.

    Enchev, R. I. et al. Structural basis for a reciprocal regulation between SCF and CSN. Cell Rep. 2, 616–627 (2012).

    Google Scholar 

  49. 49.

    Békés, M. et al. Recognition of Lys48-linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease. Mol. Cell 62, 572–585 (2016).

    Google Scholar 

  50. 50.

    Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Google Scholar 

  51. 51.

    Schrödinger, L. The PyMol Molecular Graphics System, version 1.8. (2015)

  52. 52.

    Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).

    Google Scholar 

  53. 53.

    Piana, S., Donchev, A. G., Robustelli, P. & Shaw, D. E. Water dispersion interactions strongly influence simulated structural properties of disordered protein states. J. Phys. Chem. B 119, 5113–5123 (2015).

    Google Scholar 

  54. 54.

    Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    Google Scholar 

  55. 55.

    Hornak, V. et al. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65, 712–725 (2006).

    Google Scholar 

  56. 56.

    Best, R. B., de Sancho, D. & Mittal, J. Residue-specific α-helix propensities from molecular simulation. Biophys. J. 102, 1462–1467 (2012).

    Google Scholar 

  57. 57.

    Best, R. B. & Hummer, G. Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. J. Phys. Chem. B 113, 9004–9015 (2009).

    Google Scholar 

  58. 58.

    Lindorff-Larsen, K. et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950–1958 (2010).

    Google Scholar 

  59. 59.

    Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

    Google Scholar 

  60. 60.

    Evans, D. J. & Holian, B. L. The Nose–Hoover thermostat. J. Chem. Phys. 83, 4069–4074 (1985).

    Google Scholar 

  61. 61.

    Nosé, S. A unified formulation of the constant temperature molecular dynamics methods. J. Chem. Phys. 81, 511–519 (1984).

    Google Scholar 

  62. 62.

    Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

    Google Scholar 

  63. 63.

    Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Google Scholar 

  64. 64.

    Klann, K., Tascher, G. & Münch, C. Functional translatome proteomics reveal converging and dose-dependent regulation by mTORC1 and eIF2α. Mol. Cell 77, 913–925 (2020).

    Google Scholar 

  65. 65.

    Willforss, J., Chawade, A. & Levander, F. NormalyzerDE: online tool for improved normalization of omics expression data and high-sensitivity differential expression analysis. J. Proteome Res. 18, 732–740 (2019).

    Google Scholar 

  66. 66.

    Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    Google Scholar 

  67. 67.

    Toptan, T. et al. Optimized qRT-PCR approach for the detection of intra- and extracellular SARS-CoV-2 RNAs. Int. J. Mol. Sci. 21, 4396 (2020).

    Google Scholar 

  68. 68.

    Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).

    Google Scholar 

  69. 69.

    Onafuye, H. et al. Doxorubicin-loaded human serum albumin nanoparticles overcome transporter-mediated drug resistance in drug-adapted cancer cells. Beilstein J. Nanotechnol. 10, 1707–1715 (2019).

    Google Scholar 

  70. 70.

    Corman, V. M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 25, 2000045 (2020).

    Google Scholar 

  71. 71.

    Zhang, X., Ding, L. & Sandford, A. J. Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol. Biol. 6, 4 (2005).

    Google Scholar 

  72. 72.

    Moll, H. P., Maier, T., Zommer, A., Lavoie, T. & Brostjan, C. The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types. Cytokine 53, 52–59 (2011).

    Google Scholar 

  73. 73.

    Kim, D. et al. The architecture of SARS-CoV-2 transcriptome. Cell 181, 914–921 (2020).

    Google Scholar 

  74. 74.

    Vizcaíno, J. A. et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 32, 223–226 (2014).

    Google Scholar 

  75. 75.

    Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44 (D1), D447–D456 (2016).

    Google Scholar 

Download references


We thank A. Gubas, C. Joazeiro, D. Hoeller and K. Koch for critical comments on the manuscript. We also thank Swiss Light Source (SLS) for providing special beam time for this project during the peak of the COVID-19 pandemic in Switzerland and W. Meitian and O. Vincent for providing on-site support during the data collection. We thank the Quantitative Proteomics Unit (IBCII, Goethe University Frankfurt) for support and expertise in sample preparation, LC-MS instrumentation and data analysis. This work was supported by the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177), by the Max Planck Society, by NWO (H.O. and G.J.v.d.H.v.N.) by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 789016) to B.A.S., LYSFOR2625 (DFG) to A.B. and (grant agreement no. 742720) to I.D., by the grants from Else Kroener Fresenius Stiftung, Dr. Rolf M. Schwiete Stiftung, and by internal IBC2 funds to I.D.

Author information




D.S. and I.D. conceived the project. D.S. contributed protein purification, biochemical and biophysical activity assay and structure determination. R.M. performed cell biology experiments. D.G. contributed protein purification. D.B. contributed virus infection experiments, M.W. and A.W. performed RT–qPCR measurements, K.B. performed the deneddylation assay. A.B. and G.T. designed and performed mass spectrometry experiments and analysed data. L.S. and A.R.M. performed molecular dynamics simulations. K.R. contributed to RT–qPCR materials and critical advice. P.P.G. and G.J.v.d.H.v.N. synthesized Ub(l) probes and reagents in the laboratory of H.O. S.M. and K.-P.K. provided Ubl probes and reagents. B.A.S., G.H., J.C., S.C. and I.D. supervised the project. D.S. and I.D. analysed the data and wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Ivan Dikic.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Rolf Hilgenfeld, Ingrid Wertz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Biochemical properties of SCoV2-PLpro.

a, Sequence similarity of PLpro from SARS, MERS and SARS-CoV-2. b, IFN-α treated HeLa cell lysates were incubated with PLpro for indicated time points and analysed by immunoblot c, Propargylamide-activity based probes of ubiquitin like modifiers were reacted with (left) SCoV-PLpro (right) PLproCoV2. d, ISG15-Prg were incubated with SCoV-PLpro (left) or SCoV2-PLpro (right) with increasing amount of non-hydrolysable K48-Ub2. e, Initial AMC release rate from ISG15-AMC. Purified SCoV-PLpro and SCoV2-PLpro were incubated with ISG15-AMC and indicated amounts of K48-Ub2. The release of AMC was measured by increase of fluorescence at (Ex./Em. 360/487 nm). f, Purified mUSP18 (left) and SCoV2-PLpro (right) were incubated with ISG15-propargylamide activity-based probes for indicated time points. g, Catalytic efficiency (kcat/KM) of mUSP18 and SCoV2-PLpro on ISG15-AMC cleavage. h, Sequence alignment of PLpro cleavage site of Nsp1/2, Nsp2/3, Nsp3/4 from SARS-CoV2 and human ubiquitin like modifiers. i, Hyper-NEDDylated CUL1-RBX1 was incubated with purified PLpro proteins for indicated time points at 37 °C. Reactions were performed side-by-side by with well-characterized deneddylating enzymes (DEN1 with broad specificity or COP9 Signallosome CSN specific for NEDD8 linked directly to a cullin), or the broad specificity deubiquitinating enzyme USP2 as controls. Data in e, g are presented as mean ± s.d. (n = 3, independent experiments). ** P < 0.01, *** P < 0.001, **** P < 0.0001; two-tailed paired t-tests. Experiments in b–d, f, i were repeated three times independently with similar results. Source data

Extended Data Fig. 2 Complex structure of SCoV2-PLpro with mouseISG15.

a, Structural comparison of mouseISG15:SCoV2-PLpro with humanISG15:MERS-PLpro (PDB: 6BI816) and sequence alignment of human and mouse ISG15. b, Activity test of wild type or catalytically inactive mutant (C111S) of SCoV-PLpro and SCoV2-PLpro. ISG15 Propargyl-activity based probes were mixed with indicated PLpro proteins. Experiments were repeated three times independently with similar results. c, Structural comparison of C-terminal domain of ISG15 in complex with SCoV2-PLpro and SCoV-PLpro (PDB: 5TL717). d, Snapshots from molecular dynamics simulations of SCoV2-PLpro (light pink cartoon) with (left) K48-Ub2 at 340 ns and (right) mISG15 at 3.2 μs. Key residues in the interface are highlighted. e, Backbone r.m.s.d. of the N-terminal domain of mISG15 (green) and of the distal ubiquitin in K48-Ub2 in an apo-like model (orange, model 1, SCoV2-PLpro coordinates from substrate unbound form, PDB: 6W9C) and in an mISG15-like model (yellow, model 2, SCoV2-PLpro coordinates from substrate bound form, PDB: 6YVA) from their respective SCoV2-PLpro-bound starting structures as function of time. The r.m.s.d. was calculated after superimposing the helix backbone atoms of SCoV2-PLpro. Time points for structural snapshots in e) are marked with a cross. f, Minimum heavy atom distance between F70 (SARS) and I44(Ub) in wild type and double mutant (S67V/L76T) of SCoV-PLpro:K48-Ub2 as function of time. g, Water mediated dissociation pathway. (left) Initial hydrophobic interactions between F69(CoV2), T75(CoV2) and I44(Ub). (middle) Water wedges in between T75(CoV2) and I44(Ub). (right) Water penetration between T75(CoV2)/F69 (CoV2) and I44(Ub) leads to dissociation.

Extended Data Fig. 3 Sequence alignment of papain like protease domain from corona viruses.

The amino acid sequences of papain-like protease domain from eight different coronaviruses (SARS-CoV-2, SARS, MERS, humanCoV-OC43, humanCoV-229E, humanCoV-NL63, murine HepatitisV, bovine CoV) were aligned with Clustal Omega. Accession numbers: SARS-CoV-2 (NC_045512), SARS (PDB: 3MJ5), MERS (PDB: 5W8U), hCoV-OC43 (AY585228), hCoV-229E (X69721), hCoV-NL63 (NC_005831), murine HepatitisV (NC_001846), bCoV (NC_003045).

Extended Data Fig. 4 Structural analysis of GRL-0167, SCoV2-PLpro complex.

a, Structural model of GRL-0617 bound SCoV2-PLpro. The conformation of Tyr268 on SCoV2-PLpro and the coordinates of GRL-0617 is obtained from the SCoV-PLpro:GRL-0617 structure (PDB: 3E9S18) b, Snapshots of SCoV-PLpro (light cyan) and SCoV2-PLpro (light pink) with bound GRL-0617 (dark colours) after 1 μs of molecular dynamics simulation. The protein backbones are shown in cartoon representation, and the ligand with contacting residues as sticks. c, r.m.s.d. of the GRL-0617 bound to SCoV-PLpro (light blue) and SCoV2-PLpro (light pink) as a function of time. The r.m.s.d. was calculated for non-hydrogen atoms of GRL-0617 with respect to the starting structures in the MD simulations after superimposing the helix backbone atoms of PLpro. d, In vitro PLpro inhibition assay. Initial velocity of AMC release from ubiquitin-AMC in different concentration of GRL-0617 was measured and normalized to DMSO control. IC50 of GRL-0617 to SCoV-PLpro and SCoV2-PLpro were presented. Data are presented as mean ± s.d. (n = 3, independent experiments). e, In vitro PLpro inhibition assay. Initial velocity of AMC release from ISG15-AMC in different concentration of GRL-0617 was measured and normalized to DMSO control. IC50 of GRL-0617 to SCoV-PLpro were presented. Data are presented as mean ± s.d. (n = 3, independent experiments). f, Effects of GRL-0617 on (left) deISGylase or (right) deubiquitinase activity of PLpro of SARS and SARS-CoV-2. g, Effects of GRL-0617 on SCoV-PLpro activity to (left) ubiquitin or (right) K48-Ub2 propargyl activity-based probes. Inhibitory effect of GRL-0617 on ubiquitin species was tested with various concentration of GRL-0617 (0-400 μM). h, Effects of GRL-0617 on SCoV2-PLpro activity to (left) ISG15-Cterm or (right) ISG15 propargylamide activity-based probes. Inhibitory effect of GRL-0617 on ISG15 was tested with various concentration of GRL-0617 (0-400 μM). Experiments in fh were repeated three times independently with similar results. Source data

Extended Data Fig. 5 Physiological roles of PLpro in cells.

a, b, Effect of SERPIN B3 on PLpro mediated IFN-β (a) or NF-κB p65 (b) expression level. A549 Cells were co-transfected with indicated GFP-PLpro and Myc-SERPINs and treated with either poly (I:C) or TNF-α to induce IFN-β and NF-κB p65 expression, respectively. Fold changes of luciferase level are presented. c, Effect of PLpro on IFN-induced cellular ISGylation. A549 cells were transfected with indicated PLpro plasmids and treated with IFN-α. Lysates were analysed by immune-blotting with indicated antibodies. d, e, Effect of PLpro on IFN-signalling pathway. d, A549 cells were transfected with indicated PLpro plasmids and treated with IFN-α. Lysates were analysed by immune-blotting with indicated antibodies. e, Effect of PLpro on cellular localization of IRF3. Cells from d were fractionated into cytosol and nucleus and the level of IRF3 was analysed. Lamin B1 was used for nuclear fraction control. f, Effect of PLpro on the NF-κB pathway. IκB-α phosphorylation and degradation were examined from A549 cells expressing indicated GFP-PLpro under treatment of TNF-α. g, in vitro IκBα deubiquitylation assay. Ubiquitinated IκBα were incubated with SCoV-PLpro or SCoV2-PLpro. USP2 were used as positive control. h, Effect of PLpro on NF-κB p65 cellular localization. Scale bar = 10 μm. Data in a, b, h are presented as mean ± s.d. (n = 3, independent experiments). * P < 0.05, ** P < 0.01; two-tailed paired t-tests. Experiments in ch were repeated three times independently with similar results. e, Effect of PLpro on the NF-κB pathway. IκB-α phosphorylation and degradation were examined from A549 cells expressing indicated GFP-PLpro under treatment of TNF-α. Source data

Extended Data Fig. 6 Effect of PLpro on IFN-β or NF-κB p65 expression level.

a, b, Effect of PLpro on IFN-β (a) or NF-κB p65 (b) expression level. A549 Cells were transfected with indicated GFP-PLpro and treated with either poly (I:C) or TNF-α to induce IFN-β and NF-κB p65 expression, respectively. c, d, Effect of GRL-0617 on PLpro mediated IFN-β (c) or NF-κB p65 (d) expression level. A549 Cells were transfected with indicated GFP-PLpro and treated with either poly (I:C) or TNF-α to induce IFN-β and NF-κB p65 expression, respectively. GRL-0617 is treated as indicated. All Data are presented as mean ± s.d. (n = 3, independent experiments). * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed paired t-tests. Source data

Extended Data Fig. 7 Inhibitory effects of GRL-0617 on SARS-CoV2 infection.

a, Intracellular virus production was analysed by PCR targeting SARS-CoV-2 RdRP mRNA. Relative expression level of SARS-CoV2-2 genomic RNA was normalized to cellular GAPDH level. b, Intracellular RNA was isolated from cells without infection or cells infected with SARS-CoV-2 with or without treatment of GRL-0617. Relative mRNA-level fold change of indicated genes were analysed in a qRT–PCR analysis and normalized to ACTB levels. Data in a, b are presented as mean ± s.d. (n = 3, independent experiments). * P < 0.05, ** P < 0.01; two-tailed paired t-tests. c, Schematic representation of the role of SARS-CoV-2 PLpro in the viral life cycle. The physiological role of SCoV2-PLpro in both host-immune response and polypeptide processing is shown. Inhibition of PLpro by GRL-0617 is also presented. Source data

Extended Data Table 1 Data collection and refinement statistics (molecular replacement)
Extended Data Table 2 Kinetic parameters on AMC substrates
Extended Data Table 3 Binding kinetics of PLpro to K48-Ub2 or ISG15

Supplementary information

Supplementary Figure 1

This file contains uncropped full-size gel images used in the mansucrtipt.

Reporting Summary

Peer Review File

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shin, D., Mukherjee, R., Grewe, D. et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature (2020).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing