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

Abstract

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.

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Acknowledgements

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.

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Authors

Contributions

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.

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Correspondence to Ivan Dikic.

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The authors declare no competing interests.

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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.

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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

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Shin, D., Mukherjee, R., Grewe, D. et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature (2020). https://doi.org/10.1038/s41586-020-2601-5

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