Journal: Phytochemistry

Article Title: Natural biflavones are potent inhibitors against SARS-CoV-2 papain-like protease

doi: 10.1016/j.phytochem.2021.112984

Figure Lengend Snippet: Functions of SARS-CoV-2 PL pro and benefits by targeting PL pro . (A) PL pro proteolyzes viral polyprotein at the LXGG site to generate mature nsp1, nsp2 and nsp3. (B) PL pro removes ISG15 and ubiquitin modifiers from host proteins to evade innate antiviral immunity.

Article Snippet: Recombinant SARS-CoV-2 papain-like protease (Glu1564-Val1880, Cat. 40593-V07E) and recombinant human pro-ISG15 protein (Met1-Ser165, Cat. 12729-HNAE) were purchased from Sino-Biological Inc (Beijing, China).

Techniques:

Journal: Nature

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

doi: 10.1038/s41586-020-2601-5

Figure Lengend Snippet: 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) PLpro CoV2 . d , ISG15-Prg were incubated with SCoV-PLpro (left) or SCoV2-PLpro (right) with increasing amount of non-hydrolysable K48-Ub 2 . e , Initial AMC release rate from ISG15-AMC. Purified SCoV-PLpro and SCoV2-PLpro were incubated with ISG15-AMC and indicated amounts of K48-Ub 2 . 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 ( k cat / K m ) 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.

Article Snippet: Protein sequences of the PLpro-Ubl domain of SARS and MERS (PDB ID: 3MJ5 and 5W8U, respectively , ) were also codon optimized, synthesized and cloned into pET28b with NcoI and XhoI to add C-terminal His tags (Genescript).

Techniques: Sequencing, Incubation, Western Blot, Activity Assay, Ubiquitin Proteomics, Purification, Fluorescence, Two Tailed Test

Journal: Nature

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

doi: 10.1038/s41586-020-2601-5

Figure Lengend Snippet: a , Structural comparison of mouseISG15:SCoV2-PLpro with humanISG15: MERS-PLpro (PDB: 6BI8 ) 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: 5TL7 ). d , Snapshots from molecular dynamics simulations of SCoV2-PLpro (light pink cartoon) with (left) K48-Ub 2 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-Ub 2 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-Ub 2 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.

Article Snippet: Protein sequences of the PLpro-Ubl domain of SARS and MERS (PDB ID: 3MJ5 and 5W8U, respectively , ) were also codon optimized, synthesized and cloned into pET28b with NcoI and XhoI to add C-terminal His tags (Genescript).

Techniques: Comparison, Sequencing, Activity Assay, Mutagenesis, Ubiquitin Proteomics

Journal: Nature

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

doi: 10.1038/s41586-020-2601-5

Figure Lengend Snippet: 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).

Article Snippet: Protein sequences of the PLpro-Ubl domain of SARS and MERS (PDB ID: 3MJ5 and 5W8U, respectively , ) were also codon optimized, synthesized and cloned into pET28b with NcoI and XhoI to add C-terminal His tags (Genescript).

Techniques: