Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies
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.