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

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging to characterize. Encoded within the multidomain Nsp3 is the papain-like protease PLpro that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal PLpro. Using a MALDI-TOF based assay, we screen 7538 approved clinical compounds and identify five compounds that inhibit PLpro. Despite their ability to inhibit PLpro with IC50s in the low micromolar range, the inhibitors do not have any appreciable antiviral activity in cellular SARS-CoV2 infection assays. We therefore engineered nanobodies that bind to the S1 site of PLpro with nanomolar affinity and inhibit the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology.