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.
Highlights
COVID-19 has taken a significant toll on human life and has impacted society in unprecedented ways
papain-like protease domain (PLpro) is a multifunctional enzyme with three distinct protease activities that catalyse the cleavage of (i) K48-linked polyubiquitin, (ii) ISG15 and (iii) viral polypeptides
While recent studies have characterised the substrate specificity and cleavage activity of PLproCoV-2 towards polyUb and ISG15 [7, 9, 20, 21], we wondered if elements outside the minimal PLpro domain might play a role in substrate recognition and cleavage
Summary
COVID-19 has taken a significant toll on human life and has impacted society in unprecedented ways. Understanding the mechanisms by which SARS-CoV-2 interacts with human cells and the functions of the different viral proteins in establishing infection is of paramount importance. The genome of SARS-CoV-2 encodes 16 non-structural proteins (Nsps) which play different roles in the viral life cycle [1]. The largest of the Nsps, is a key component of the viral replication and transcription complex assembled on host cell membranes where replication and transcription of the viral genome occurs [2,3,4]. Another essential role of Nsp is its function as a protease. While several coronaviruses express two PLpro enzymes, SARS-CoV-2 only expresses one PLpro enzyme [1, 5]
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