The papain-like protease determines a virulence trait that varies among members of the SARS-coronavirus species
Fig 2
SR-PLP had conserved protease cleavage-, DUB- and deISGylating activities.
(a) HEK-293T cells were transfected with empty vector plasmid (EV) or plasmids expressing either wild type (WT)- or catalytic mutant (CA) PLPs. SARS-CoV nsp2/3-GFP was cotransfected simultaneously. Lysates were harvested at 16 hours post transfection (hpt), and gene expression was analyzed by Western blotting. (b) A biosensor assay was applied for the detection of PLP cleavage activity. Cells were cotransfected with pGlo Firefly luciferase, and either WT-PLP, CA or EV plasmids in 96-wells. At 14 hpi, cells were incubated with GloSensor reagent and luminescence was detected. (c) To investigate the activity spectrum of a SA-PLP protease inhibitor, one hour after the GloSensor incubation, 12.5, 25 or 50 μM of compound 3e or DMSO were added. PLP activities were analyzed in relation to the different amounts of compound 3e at 4 h post treatment. Values were normalized to the respective DMSO-treated WT-PLP. Biosensor assays were performed in triplicate and repeated three times independently. Error bars indicate standard deviations of the means. Statistical significance between DMSO and inhibitor-treated cells or cells transfected with the CA-PLPs, respectively, was determined using one-way ANOVA and Sidak post hoc test. Statistically significant differences are indicated by asterisks (p> 0.05 not significant (ns), p≤ 0.05 significant (*), p≤ 0.01 very significant (**), p≤ 0.001 highly significant (***)). (d) HEK-293T cells were transfected with EV or plasmids expressing either WT- or CA-PLPs. For analysis of DUB activity WT-PLP plasmids were transfected in increasing amounts of plasmids (50 ng, 100 ng and 200 ng per 12-well). Control plasmids (200 ng) EV and CA-PLP were transfected for comparison. HA-ubiquitin (HA-ub) was coexpressed in all samples. Lysates were harvested at 18 hpt, and gene expression was analyzed by Western blotting. (e) For analysis of deISGylating activity pISG15-myc and the conjugation machinery (UbcH8, Ube1L, and Herc5) of ISG15 were coexpressed in all samples. Lysates were harvested at 18 hpt, and gene expression was analyzed by Western blotting. Western blot experiments were repeated three times independently and one representative blot is shown. β-Actin was applied as loading control.