Characterization of protease activity of Nsp3 from SARS-CoV-2 and itsin vitroinhibition by nanobodies

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 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.

Integrative multi-omics landscape of non-structural protein 3 of severe acute respiratory syndrome coronaviruses

“…To meaningfully study the functions of NSP3, it needs to be expressed in its full-length form in mammalian cells. Recent reports demonstrated that PLpro alone is not sufficient to cleave the NSP1-NSP2 polypeptide, and the PLpro domain has a very similar interactome with NSP3 without the transmembrane region, suggesting that the localization and integrity of the NSP3 are important for its function and interaction with host cells [18,21]. A previous study using deletions showed that CoV2-NSP3 has a general cytosolic localization, which is contradictory to our results [50].…”

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease

“…Over the 20 min of incubation, increasing fluorescence signal was observed for substrate Pro2 but not for Pro1 (Supplementary Figure S2A). Consistent with this, it has been recently shown that full-length nsp3 protein but not the isolated PLpro domain can cleave between nsp1/2 [18]. Although cleavage of Pro2 occurred, the rate was relatively slow making it less suitable for screening.…”

“…We performed orthogonal assays using two different substrates of PLpro to test the inhibition of isopeptidase activity of PLpro by the small molecule inhibitors identified in the screen. When K48-linked triubiquitin (Ub3) is incubated with PLpro, it is efficiently cleaved to diubiquitin (Ub2) and monoubiquitin (Ub1) as the final products [ 18 ]. Similarly, pro-ISG15 is cleaved to ISG15.…”

“…We have found that dihydrotanshinone I is the best inhibitor of SARS-CoV-2 PLpro and did not inhibit 3CLpro. Although we, like other groups [ 24 , 27 , 28 ], used a truncated nsp3 protein including just the Ubl2 and PLpro domains, and there is evidence that cleavage by the full-length nsp3 may have slightly different specificities [ 18 ]. The fact that dihydrotanshinone I stops viral replication suggests that it is a good nsp3 PLpro inhibitor in cells.…”

“…The deubiquitinating (DUB) and deISGylating activities of PLpro following LXGG motifs thus have an implication in viral invasion by shutting down the viral-induced host innate immune response [ 30 ]. PLpro of SARS-CoV-2 further shows preferential activity in cleaving ISG15 over Ub in comparison with SARS-CoV-1 [ 18 , 22 , 24 ]. Interestingly, although the high transmission (and potentially more deadly) U.K. variant of SARS-CoV-2, B.1.1.7 was believed attributed to multiple mutations in the spike protein, the significance of a particular point mutation, A1708D, in PLpro for viral invasion remains unexplored [ 31 ].…”