Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro 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 Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain 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 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.

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Author:Lee A. ArmstrongORCiD, Sven M. LangeORCiD, Virginia De CesareORCiD, Stephen P. MatthewsORCiD, Raja Sekar NirujogiORCiD, Isobel Cole, Anthony Hope, Fraser CunninghamORCiD, Rachel Toth, Rukmini MukherjeeORCiD, Denisa BojkovaORCiDGND, Franz Sebastian GruberORCiDGND, David GrayORCiD, Paul G. WyattORCiD, Jindrich CinatlORCiDGND, Ivan ĐikićORCiDGND, Paul Davies, Yogesh KulathuORCiDGND
Parent Title (English):bioRxiv
Document Type:Preprint
Date of Publication (online):2020/12/09
Date of first Publication:2020/12/09
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/11/19
Page Number:45
Angeschlossene und kooperierende Institutionen / MPI für Biophysik
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International