Sars cov 2 plpro

Recombinant SARS-CoV-1 PL^pro, SARS-CoV-2 PL^pro and Ubiquitin-AMC were purchased as 32, 11 and 250 μM solutions, respectively, from R&D Systems.
Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one) is commercially available from Sigma Aldrich.
All compounds were obtained and fully characterized in previous studies^{39 (link)}. Their purity and homogeneity were confirmed by HRMS and ⁷⁷Se NMR (see Supplementary data).
Enzyme and inhibition assays were designed based on the procedures described in^{14 (link),16 (link),39 (link),40 (link)}.

Supernatant from SARS-CoV-2 and mock infected Calu-3 cells was combined with the PLpro substrate, Ubiquitin-Rh110 (R&D Systems, #U-555) at 670 nM in phenol-free RPMI. Fluorescence was measured at 535nM on a plate reader. Samples were run in duplicates. A standard curve was generated by measuring protease activity from recombinant SARS-CoV-2 PLpro (R&D) diluted in mock infection medium at 1, 10, 100, 500, and 1000nM and run on the same plate as experimental samples.

Neuronal Ca²⁺ imaging experiments were carried out as previously described.^{61 (link)} Neuronal cultures were loaded for 45 min at room temperature with 10 μM Fura-2AM supplemented with 0.01% Pluronic F-127 (wt/vol, Life Technologies) in a physiological Ringer’s solution containing (in mM) 140 NaCl, 5 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2 and 10 D-(+)-glucose, pH 7.4. Cells were identified as neurons by eliciting depolarization with high potassium Ringer’s solution (75 mM) at the end of each experiment. Acquired images were displayed as the ratio of 340 nm / 380 nm. Fura-2 ratios were normalized to baseline ratio. Responding neurons were defined as those having a > 10% increase from baseline ratio. Human iPSC-derived neurons were defined as responding to > 5% increase from baseline ratio. All calcium imaging analyses were performed using custom python scripts. Neurons were stimulated with SARS-CoV-2 PLpro (R&D #E-611), SARS-CoV PLpro (R&D #E-610), MERS-CoV PLpro (R&D #E-609), SARS-CoV-2 PLpro (Cayman #31817) diluted in Ringer’s to the appropriate concentration. Vehicle (50 mM HEPES, 300 mM NaCl, 1 mM TCEP, 10% (v/v) Glycerol, pH 8.0) dilution was matched to that of PLpro. Cells were incubated in the TRPA1 antagonist (HC-030031, Tocris #2896) for 15 minutes prior to imaging experiment.