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

PLoS One. 2021 Jul 16;16(7):e0253364. doi: 10.1371/journal.pone.0253364. eCollection 2021.

Abstract

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 Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. 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 catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Antigen-Antibody Complex
  • Antiviral Agents / pharmacology*
  • Humans
  • Inhibitory Concentration 50
  • Protease Inhibitors / pharmacology*
  • RNA-Dependent RNA Polymerase / antagonists & inhibitors*
  • RNA-Dependent RNA Polymerase / immunology
  • RNA-Dependent RNA Polymerase / metabolism
  • Single-Chain Antibodies / immunology
  • Single-Chain Antibodies / pharmacology*
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Viral Nonstructural Proteins / immunology
  • Viral Nonstructural Proteins / metabolism

Substances

  • Antigen-Antibody Complex
  • Antiviral Agents
  • Protease Inhibitors
  • Single-Chain Antibodies
  • Viral Nonstructural Proteins
  • Nsp3 protein, SARS-CoV
  • RNA-Dependent RNA Polymerase