Fig. 6: Identification of substrates deISGylated by SARS-CoV-2 PLpro.

a, Coomassie-stained gel of fractions collected from size-exclusion chromatography for bacterially expressed and purified WT and mutant (C111A) SARS-CoV-2 histidine-tagged PLpro. b, Cell lysates from type I IFN-treated macrophages or HeLa cells were treated with buffer alone (control lanes), purified WT or mutant (C111A) PLpro. Lysates were resolved by gel electrophoresis and visualized by western blotting. c, Activity-based assay to determine deubiquitylating versus deISGylating activities of WT and C111A PLpro; 10 µM of either HA-tagged Ub-PA or ISG15-PA were treated with the indicated concentrations of WT and mutant (C111A) PLpro for 30 min at room temperature. USP18 was used as a positive control for deISGylase activity. Reaction products were resolved by gel electrophoresis and visualized by Coomassie staining. d, Sample preparation for mass spectrometry to identify PLpro substrates. Western blot analysis of cellular lysates obtained from HeLa WT or ISG15^−/− cells after 72 h of type I IFN stimulation and treatment with recombinant WT or mutant PLpro for 30 min. The same lysates were used for the actual ISG15-GlyGly peptidomics experiment. ISG15 and ubiquitin conjugates were visualized by immunoblotting with anti-ISG15 (IB:ISG15) and anti-ubiquitin (IB:UBQ) antibody, respectively. Tubulin-α served as a loading control and was detected with anti-tubulin-α antibody (IB:Tub-α). a–d, Images are representative of three biologically independent experiments. e, Heatmap showing significantly regulated GlyGly(K) sites after unsupervised hierarchical clustering. Different genotypes (WT or ISG15 knockout) and protease treatments (WT or mutant PLpro) are indicated. The colors indicate upregulated (red) or downregulated (blue) sites. Right, The same heatmap is shown with the originally missing values depicted in gray. Three major clusters can be observed that contain the ISG15 sites targeted (cluster 1a/1b) or untargeted (cluster 2) by PLpro, or ubiquitin sites unaffected by PLpro treatment (cluster 3). f, Validation of identified hits in macrophages expressing either WT or C111A mutant variants of PLpro, radiolabeled with [³⁵S]cysteine/methionine and endogenous proteins immunoprecipitated with antibodies against the indicated proteins. Autoradiographs are representative of three biologically independent experiments.