Fig 1.
Nsp3core is a more active protease compared to PLpro.
A) Schematic of cleavage of Nsp1-3 by PLpro from the viral polypeptide and the domain structure of Nsp3: Ubl (ubiquitin-like domain), ADRP (ADP-ribose phosphatase, SUD (SARS-unique domain), PLpro (papain-like protease), NAB (nucleic acid binding domain), TM (Transmembrane domain), ZnF (zinc finger motif). B) Coomassie-stained gel of Nsp3 constructs used in this study with accompanying domain schematics (colour coding as in 1A; double slash indicates deletion). C) DUB assay directly comparing cleavage of Ub3 (upper) and ISG15 (lower) by PLpro and Nsp3core. D) Assay directly comparing cleavage of Nsp1-2Δ by PLpro and Nsp3core. E) Protease assay comparing the cleavage of Nsp1-2 FL by PLpro and Nsp3core. F) DUB assay showing the polyubiquitin linkage preference of Nsp3core. G) Cleavage assay comparing cleavage of Nsp1-2Δ and Nsp1-2Δ G180A mutant by Nsp3core. H) UV-traces of analytical gel filtration analyses (Superdex 200 3.2/300) of Nsp3core overlaid onto molecular weight standards. I) Mass photometry analysis measuring the molecular mass of Nsp3core. Data shown are representative of two independent experiments.
Fig 2.
Comparison of Nsp3 and PLpro interactome.
Volcano plot showing significant interactors identified by pull down on Nsp3 DTM or PLpro from transiently transfected cells followed by quantitative mass spectrometry. Interactors enriched in PLpro pull downs are highlighted in blue and those enriched in Nsp3 pull downs are highlighted in red circles.
Fig 3.
Identification and characterization of PLpro inhibitors.
A) Schematic workflow of the MALDI-TOF DUB assay. K48 trimer is incubated with PLpro for 30 minutes at room temperature. The reaction is stopped by adding 2% TFA and 15N ubiquitin (as internal standard). Enzymatic activity and inhibition were assessed by MALDI-TOF MS detection of ubiquitin and 15N ubiquitin signals. B) FDA approved compound library screening by MALDI-TOF, Z’Prime score and HTS data distribution. C) IC50 calculation of compounds returning >50% inhibition values in the HTS: thioguanine, nordihydroguaiaretic acid, Disulfiram, Auranofin and Tideglusib. IC50s were calculated both against PLpro and Nsp3core using the MALDI-TOF DUB Assay. D-E) Orthogonal, gel-based assays testing PLpro and Nsp3 protease activities against K48 trimer and pro-ISG15 as substrates. Nordihydroguaiaretic acid (NDA), Disulfiram (DSF), Auranofin (AUR) and Tideglusib (TID) were tested at the indicated concentrations against PLpro and Nsp3core. Data shown are representative of two independent experiments. F) IC50 calculation of GRL0617 against PLpro and Nsp3core. G) Selectivity assessment of GRL-0617 and HTS positive hits against a panel of 42 human DUBs and the viral ovarian tumor (vOTU). Compounds were tested at the final concentrations of 10 and 1 μM.
Fig 4.
Development of nanobodies that inhibit Nsp3core.
A) Schematic workflow of yeast-surface display selection to identify site-specific nanobodies as competitive Nsp3core inhibitors. B) Flow cytometry analysis of yeast displaying anti-Nsp3 nanobodies NbSL17, NbSL18 or NbSL19, respectively. Yeast were stained with anti-HA-Alexa488 against HA-tagged Nb and Streptavidin-Alexa647 against biotinylated antigens. Top: Yeast incubated with PLpro wild-type, Bottom: Yeast incubated with PLpro S1*-mutant. Percentages indicate fraction of yeast positive for both fluorescent labels. C) UV-traces and coomassie-stained SDS-PAGE of analytical gel filtration analyses of PLpro-Nb complexes. PLpro alone (black), PLpro+NbSL17 (orange) and PLpro+NbSL18 (blue). D) Isothermal calorimetry titration measuring binding of PLpro to NbSL18. E) Silver stained gels of DUB assays of PLpro (left) and Nsp3core (right) with K48-Ub3 (top) and proISG15 (middle) and Nsp1-2 FL (bottom) as substrates in the presence of increasing concentrations of NbSL18. Silver-stained SDS-PAGE gels shown are representative of two independent experiments. Also see S3 Fig.