The papain-like protease domain sequence is obtained from SARS-CoV-2 complete genome (NCBI genome databank, Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu- 1, complete genome; NC_045512). Protein sequence for PLproCoV2 domain (amino acids, 746- 1060) of Nsp3 protein from SARS-CoV-2 (Nsp3; YP_009725299.1) was codon optimized, synthesized and cloned into pET28b with NcoI and XhoI to have C-terminal His-tag (Genescript). Protein sequences of PLpro domain of SARS and MERS from (PDB ID: 3MJ5, 5W8U, respectively) were also codon optimized, synthesized and cloned into pET28b with NcoI and XhoI to have C-terminal His-tag (Genescript). Mutants were generated by PCR and verified with sequencing. For mammalian expression, PLpros are cloned into pEGFP-C1 (clontech).
BL21(DE3) E. coli competent cells (NEB) were transformed with plasmids and grown in LB medium to an OD600 of 0.6-0.8 at 37°C. Recombinant murine ISG15 were kindly gifted from Dr. K.P. Knobeloch (University Freiburg). Protein expression was induced by addition of 0.5 mM IPTG (isopropyl D-thiogalactopyranoside) and 1 mM Zinc Chloride (ZnCl2) the cells were further grown overnight at 18°C and harvested. The cell pellet was resuspended in lysis buffer 50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, 2 mM DTT, pH 8.5) and lysed by sonication and centrifuged at 13,000 rpm to clarify the supernatant. The supernatant was incubated 2 hours with TALON beads (Takara) pre-equilibrated with lysis buffer and non- specific proteins were cleared with washing. Proteins were eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, 2 mM DTT, pH 8.5). Eluted proteins were buffer exchanged to storage buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, pH 8.5) and stored for biochemical analysis. For crystallization of PLproCoV2-C111S, the cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, 1 mM TCEP, pH 7.4) and lysed by sonication and centrifuged at 13,000 rpm to clarify the supernatant. The supernatant was incubated 2 hours with TALON beads (Takara) pre-equilibrated with lysis buffer and non-specific proteins were cleared with washing. Proteins were eluted with elution buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, 1 mM TCEP, pH 7.4) and further purified on size-exclusion column (Superdex 75 16/60, GE Healthcare) pre-equilibrated with 20 mM Tris-HCl, 100 mM NaCl, 1 mM TCEP, pH7.4. Proteins were concentrated to 20 mg/ml and stored for crystallization.
Ubiquitin/NEDD8/SUMO-/ISG15 activity-based probes assay
PLPros were diluted (2 µM, final concentration) with activation buffer and incubated 10 minutes at 25°C and the activity-based probes were diluted (0.2 mg/ml, final concentration) in dilution buffer (50 mM Tris-HCl 7.5, 150 mM NaCl). The reaction mixture was prepared by mixing equal volume of activated PLpros (2 µM) and activity-based probes (0.2 mg/ml). Reactions were conducted at indicated temperature (on ice or 37°C) and samples were taken at the indicated time points and the reactions were quenched by the addition of SDS-sample buffer. Samples were further analyzed by SDS-PAGE and stained with silver staining kit (Thermo Fisher).
The equal amount of PLproCoV2-C111S were mixed with full-length murine ISG15. Final concentration of mixture was 250 µM. Protein mixture were screened with sitting drop matrix screens in 96-well plate with 100 nl of protein and 100 nl of precipitant solution at 293K. Initial crystals appeared from solution containing 20 % PEG 3350, 200 mM Potassium thiocyanate with 250 µM protein concentration. Diffraction-quality crystals were grown in optimized solution containing 18 % PEG 3350, 100 mM bis-tris propane pH 6.5, 200 mM Potassium thiocyanate with 200 µM protein concentration.
Data collection, processing and structure determination
Crystals were cryo-protected using mother liquor solution supplemented with 25 % (v/v) ethylene glycerol. Diffraction data were collected on single frozen crystal in a nitrogen stream at 100 K at beamline PXI as Swiss Light Source, Villigen. Initial data sets were processed using XDS 42, and phases were determined by Phaser molecular replacement in ccp4 module with PLproCoV2, mISG15 as template model 43, PDB IDs: 6W9C and 5TLA, respectively). Structure refinement and manual model building were performed with Coot and Phenix.Refine 44,45.
Cell lysates deubiquitination and deISGylation assay
HeLa or A549 cells were treated with IFN-⍺ (200 U/ml) for 48 hrs to induce ISGylation. Cells were lysed with lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) NP-40) and concentration was measured with BCA assay (Thermo Fischer). 10 µg of lysates were incubated with 100 nM of PLpro for indicated timepoints at 37°C and analyzed by immunoblotting with indicated antibodies. To test inhibitory effect of GRL-0617, 40 µM of GRL-0617 was included during reaction.
Deneddylation and IkBa deubiquitination assay
All proteins described are of human origin. CUL1-RBX1, SKP1-b-TRCP2, UBE2M, UBE2D3, NEDD8, UB, APPBP1-UBA3, UBA1 were purified as previous described 46. Neddylated CUL1-RBX1 was generated as previous described 46. Reaction for generating hyperneddylated CUL1-RBX1 was driven at pH 8.8 at 37°C for 30 min, and purified size exclusion chromatography. USP2 catalytic core was purified with nickel affinity chromatography, liberated of the His-tag by overnight thrombin cleavage, following IEX and size exclusion chromatography. CSN was purified as previous described 47. Deneddylation assays were performed with 1 µM hyperneddylated CUL1-RBX1, and 5µM protease (PLproSARS, PLproCoV2, DEN1, USP2) or 20 nM CSN. Reaction was performed at 37°C in 2 5mM Tris 100 mM NaCl, 5 mM DTT pH 8.5, and in the case of CSN with additional 10mM MgCl2. Samples were taken each indicated time points and quenched with 2X SDS-PAGE sample buffer. Gels were stained by coomassie-blue and scanned on an Amersham imager 600. IkBa Deubiquitylation assays were performed by first generating a ubiquitylated IkBa, with 200 nM UBA1, 1 µM UBE2D3, 20 µM UB, 500 nM neddylated CRL1β-TRCP, and 5µM fluorescently labelled IkBa at 37°C in 50 mM Tris 50mM NaCl 10mM MgCl2 5mM DTT pH 7.5 for 30 minutes. Reaction was quenched by adding 80mM EDTA for 5 minutes. Deubiquitylation reaction was started by mixing 3 µM of protease (PLproSARS, PLproCoV2, USP2) with the ubiquitylation reaction, and samples were taken each time points and quenched with 2X SDS- PAGE sample buffer. Gels were scanned on an Amersham Typhoon (GE) detecting the fluorescently labelled IkBa.
Molecular Dynamics Simulations
PLproCoV2 with K48-Ub2:
For the model of PLproCoV2 with bound K48-Ub2, we combined the X-ray crystal structure of the apo form (PDB ID: 6W9C, re-refined by Tristan Croll (https://drive.google.com/drive/folders/1JBo50CdkBU7K1pFThuqrzhQ-NcsIAWyG) with the substrate coordinates taken from PDB ID: 5E6J48 after PLpro alignment using PyMol 49. The triazole linker was replaced with Lys using MODELLER 50. To mimic the linker, a harmonic distance restraint potential was applied between the backbone carbonyl carbon atoms of Lys48 and Gly75 with a target distance of 9.5 Å and a force constant of 502080 kJ mol−1 nm−2. The covalent propargylamide linker was removed.
PLproSARS and PLproCoV2 in complex with inhibitor GRL-0617:
The coordinates of the PLproSARS complex were taken from PDB ID: 3E9S 18. The oxidized Cys112 was changed to the reduced form (SH) using MODELLER. The simulation model of the PLproCoV2 complex was built according to the X-ray structure of the apo form (PDB ID: 6W9C, re-refined by Tristan Croll). The coordinates of the compound GRL-0617 were modeled according to PDB ID: 3E9S after PLpro alignment using PyMol. The loop (GNYQCGH) capping the GRL-0617 binding site was remodeled according to the PLproSARS X-ray crystal structure of the complex (PDB ID: 3E9S) using MODELLER. The GRL-0617 ligand was parameterized with the General Amber Force Field (GAFF) 51.
PLproCoV2 with mISG15:
The X-ray crystal structure PLproCoV2:mISG15 served as starting point. Missing residues of PLproCoV2 and one Zn ion were modeled according to the apo X-ray crystal structure (PDB ID: 6W9C, re-refined by Tristan Croll). Missing side chains in all setups were modeled using MODELLER. All crystallographic water molecules and ions were retained, except a nickel ion in PDB ID: 5E6J. According to pKa calculations using PropKa and additional visual inspections, in all setups His17CoV2 (His18SARS) and His272 CoV2 (His273SARS) were charged. All other residues were simulated in their physiological protonation state. The proteins were solvated in TIP4P-D water 52 with 150 mM NaCl. MD simulations were carried out using Gromacs 2018 53 and the AMBER99SB*-ILDN-q force field 54–57. Each system was energy minimized, followed by five equilibration steps, in which we gradually weakened the position restraints on heavy atoms, first in an NVT ensemble (0.25 ns) and then in an NPT ensemble (4 x 0.5 ns) using a Berendsen thermostat and barostat 58. Production simulations of 1 µs each were run at a temperature of 310 K and a pressure of 1 bar in an NPT ensemble using a Nosé- Hoover thermostat 59,60 and a Parrinello-Rahman barostat 61. For simulations with bound substrates and with bound inhibitor, we monitored the root-mean-square deviation (RMSD) of each backbone substrate (distal ubiquitin in K48-Ub2 and N-terminal domain of mISG15) and of GRL-0617 (heavy atoms) to the respective equilibrated structure after alignment on the helix backbone of PLpro (without the flexible UBL domain).
Inhibitor IC50 determination
For IC50 value for inhibitors, ubiquitin-AMC was used as substrate of PLpro and the release of AMC was measured by increase of fluorescence (Ex./Em. 360/487 nm) on 384-well microplate reader (PHERAstar FSX, BMG Labtech). 5ul of solution containing different concentration of GRL-0617 (200 – 0 µM) and 10 µM of ubiquitin-AMC were aliquoted into 384 well plate and reaction was initiated by addition of 5 µl of PLpro (30 nM) to the well. Initial velocities of AMC-release were normalized against to DMSO control. IC50 value is calculated by Dose- response – Inhibition function in Graphpad Prism with [inhibitor] vs normalized response equation. The experiment was repeated three times.
For interactome analysis, A549 cells were transfected with CoV2 mut PLpro/wt and for comparison between SARS and SARS-CoV-2, mutant PLpro versions for both proteins were transfected. Cells were stimulated with Interferon-⍺ (200 units/ml) for 36 hours to mimic infection scenario. Cells were lysed in ice cold lysis buffer (50 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% Triton x-100) and equal amounts of lysates were incubated with GFP nanotrap beads in IP buffer (Lysis buffer without detergent). After incubation, IPs were washed three times with wash buffer (50 mM Tris-Cl, pH7.5; 400 mM NaCl; 0.5 mM EDTA) and two times with IP buffer. Then beads were incubated with 25 ul of 50 mM tris-HCl (pH 8.5) containing 4 M urea, 1 mM Tcep, 4 mM Chloroacetamide for 1 hour in dark at 37°C. Afterwards, samples were then diluted with 50 mM Tris-cl pH 8.5 to final urea conc. < 2M and digested with 0.5 µg Trypsin (Promega) at 37°C overnight. Digests were acidified using trifluoroaceticacid (TFA) to a pH of 2-3 and peptides were enriched using stage tips 62. For getting quantitative information, peptides were labelled with TMT reagents (Thermo fisher) as described previously63. Briefly, peptides were resuspended in TMT labelling buffer (0.2 M EPPS pH 8.2, 20% Acetonitrile) and were mixed with TMT reagents in a 2:1 TMT : peptide ratio. Reaction was performed for one hour at RT and subsequently quenched by addition of hydroxylamine to a final concentration of 0.5% at RT for 15min. Samples were pooled in equimolar ratio, acidified, and cleaned up using Empore C18 (Octadecyl) resin material. After drying, peptides were resuspended in 0.1% FA for LC-MS. All mass spectrometry data was acquired in centroid mode on an Orbitrap Fusion Lumos mass spectrometer hyphenated to an easy-nLC 1200 nano HPLC system with a nanoFlex ion source (ThermoFisher Scientific). A spray voltage of 2.6 kV was applied with the transfer tube heated to 300°C and funnel RF set to 30%. Internal mass calibration was enabled (lock mass 445.12003 m/z). Peptides were separated on a self-made 32 cm long, 75 µm ID fused-silica column, packed in house with 1.9 µm C18 particles (ReproSil-Pur, Dr. Maisch) and heated to 50°C using an integrated column oven (Sonation). HPLC solvents consisted of 0.1% Formic acid in water (Buffer A) and 0.1% Formic acid, 80% acetonitrile in water (Buffer B). Peptides were eluted by a non-linear gradient from 7 to 40% B over 90 minutes followed by a step-wise increase to 95% B in 6 minutes which was held for another 9 minutes. Full scan MS spectra (350-1400 m/z) were acquired with a resolution of 120,000 at m/z 200, maximum injection time of 100 ms and AGC target value of 4 x 105. The 20 most intense precursors per full scan with a charge state between 2 and 5 were selected for fragmentation (“Top 20”), isolated with a quadrupole isolation window of 0.7 Th and fragmented via HCD applying an NCE of 38%. MS2 scans were performed in the Orbitrap using a resolution of 50,000 at m/z 200, maximum injection time of 86ms and AGC target value of 1 x 105. Repeated sequencing of already acquired precursors was limited by setting a dynamic exclusion of 60 seconds and 7 ppm and advanced peak determination was deactivated. MS raw data was analysed with Proteome Discoverer (PD, version 2.4, ThermoFisher Scientific) using Sequest HT as a search engine and performing re-calibration of precursor masses by the Spectrum RC-node. Fragment spectra were searched against the human reference proteome (“one sequence per Gene”, 20531 sequences, version March 2020) and protein sequences for SARS (15 sequences, version March 2020), CoV2 (14 sequences, version February 2020) and MERS (10 sequences, version April 2020) downloaded from Uniprot in March 2020 as well as common contaminants as included in “contaminants.fasta” provided with the MaxQuant software. Static modifications were TMT at the peptide N-terminus and lysines as well as carbamidomethyl at cysteine residues, dynamic modifications were set as Oxidation of Methionine and Acetylation at the protein-N-term. Matched spectra were filtered with Percolator applying a false discovery rate of 1% on PSM and protein level. Reporter intensities were normalised to the total protein intensities in PD assuming equal sample loading and additionally by median-normalisation using the NormalyzerDE package 64. Statistically significant changes between samples were determined in Perseus (version 22.214.171.124) using a Two-sample T-test with a Benjamini-Hochberg FDR of 5% on log2 transformed values 65.
Human CaCo–2 cells were obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). Cells were grown at 37°C in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and containing 100 IU/ml penicillin and 100 μg/ml streptomycin.
SARS-CoV–2/FFM1 was isolated from asymptomatic travellers returning from Wuhan (China) to Frankfurt (Germany) using CaCo–2 cells. SARS-CoV–2/FFM1 stocks used in the experiments had undergone one passage on CaCo–2 cells. Virus titers were determined as TCID50/ml in confluent cells in 96-well microtiter plates.
Antiviral and cytotoxicity assays
Confluent layers of CaCo–2 cells in 96-well plates were infected with SARS-CoV–2/FFM1 at MOI 0.01. Virus was added simultaneously with GRL-0617 and incubated in MEM supplemented with 1% FBS with different drug dilutions. Cytopathogenic effect (CPE) was assessed visually 48 h after infection. To assess effects of GRL-0617 on CaCo–2 cell viability, confluent cell layers were treated with different drug concentration. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-549 diphenyltetrazolium bromide (MTT) assay modified after Mosman 66, as previously described 67. Data for each condition was collected for at least three biological replicates.
Luciferase activity assay
To analyze the induction of IFNβ induced genes, a luciferase reporter assay was used in A549 cells. Briefly, an expression construct containing the luciferase ORF and the IFNβ promoter (IFNβ/luciferase) was co-transfected with either a GFP control plasmid or the designated PLpro plasmid. For all transfections, 100 ng of luciferase plasmid, 400ng of Plpro or GFP vector was used in each well of a 12 well plate All transfections were performed in triplicate and the average of 3 experiments is shown in figures. 24h post transfection cells were treated with 500ng poly I:C for 18h or 50ng/ml of TNFα for 30min. Luciferase expression was measured Luciferase Reporter assay system (Promega Inc). Fold change is calculated by taking vector treated with poly I:C or TNF-α as 1.
Immunofluorescence and confocal imaging
HeLa cells expressing GFP tagged PLPro was treated with TNFα (50ng/ml) for 45min. Cells were fixed with paraformaldehyde, blocked in 5% serum and immunostained overnight at 4°C with antibody against p65. Confocal imaging was performed using the Zeiss LSM780 microscope system. An Ar-ion laser (for excitation of GFP at 488 nm), a He-Ne laser (for excitation Alexa Fluor 546nm) were used with a 63×1.4 NA oil immersion objective. Images were analysed in FIJI to check for colocalisation between DAPI and immunostained p65. Results are indicative of 50 cells taken from 3 independent experiments; error bars indicate standard deviation.
A549 cells from a confluent 60 mm dish were transiently transfected with GFP tagged PLPro followed by treatment with interferonα (200u/ml, 36h). Cells were lysed in hypotonic buffer [10 mmol/L HEPES (pH = 7.4), 2 mmol/L MgCl2, 25 mmol/L KCl, 1 mmol/L DTT, 1 mM PMSF, and protease inhibitor cocktail], kept on ice for 30 min followed by syringe lysis, 2 mol/L sucrose solution was added dropwise, followed by centrifugation at 1000g for 15 min. The supernatant was saved as the cytosolic fraction. The pellet was washed twice in wash buffer [10 mmol/L HEPES (pH = 7.4), 2 mmol/L MgCl2, 25 mmol/L KCl, 250 mmol/L sucrose, 1 mmol/L DTT, 1 mmol/L PMSF, and protease inhibitor cocktail] and saved as the nuclear fraction.
Quantification of viral and cellular RNA
SARS-CoV-2 RNA from cell culture supernatant samples was isolated using ACL buffer and the QIAamp 96 Virus kit (Qiagen) according to the manufacturer’s instructions. RNA was subjected to OneStep qRT-PCR analysis using the LightCycler Multiplex RNA Virus Master kit (Roche). Intracellular RNA was isolated using RLT buffer and the RNeasy 96 HT Kit according to the manufacturer’s instructions. PCR was performed on a CFX96 Real-Time System, C1000 Touch Thermal Cycler. Primers and probe were adapted from the WHO protocol 68 targeting the open reading frame for RNA-dependent RNA polymerase (RdRP) of both SARS-CoV-2: RdRP_SARSr-F2 (GTGARATGGTCATGTGTGGCGG) andRdRP_SARSr-R1 (CARATGTTAAASACACTATTAGCATA) primers were used in a final concentration of 0.4 µM and RdRP_SARSr_P2 probe (6-Fam CAGGTGGAACCTCATCAGGAGATGC BBQ1) was used with 0.2 µM, respectively. Primers for ACTB (fwd:CATCGAGCACGGCATCGTCA; rev:TAGCACAGCCTGGATAGCAAC) 69 and ISG15 (fwd: GAGAGGCAGCGAACTCATCT; rev: AGGGACACCTGGAATTCGTT) 70 were used for SYBR green based detection of cellular genes in a final concentration of 0.4 µM per reaction. For each condition, three biological replicates were used. Mean and standard deviation were calculated for each group.