Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies
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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 IC 50 s 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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SciScore for 10.1101/2020.12.09.417741: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources pET15D Twinstrep 3C SARS-CoV-2 Nsp3 N179-N1329 6His C856A (DU67898) was made by adding the C856A mutation to clone DU67831 using PCR mutagenesis. C856Asuggested: NoneTransfections, lysis and Co-Immunoprecipitations: A549 human alveolar epithelial cells were transfected with empty vector in triplicate or with vectors expressing SARS CoV-2 Nsp3ΔTM or PLpro fused to a 2xStrep tag at the N-terminus (4 replicates each). A549suggested: …SciScore for 10.1101/2020.12.09.417741: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources pET15D Twinstrep 3C SARS-CoV-2 Nsp3 N179-N1329 6His C856A (DU67898) was made by adding the C856A mutation to clone DU67831 using PCR mutagenesis. C856Asuggested: NoneTransfections, lysis and Co-Immunoprecipitations: A549 human alveolar epithelial cells were transfected with empty vector in triplicate or with vectors expressing SARS CoV-2 Nsp3ΔTM or PLpro fused to a 2xStrep tag at the N-terminus (4 replicates each). A549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)Experimental Models: Organisms/Strains Sentences Resources pTXB1 Halo 3C SARS-CoV-2 (2019-nCoV) Nsp1 1-179-Intein CBD (DU67780 was made using NEBuilder (New England Biolabs), amplifying the vector from existing clone DU28033 (pTXB1-HALO-Mxe Intein-CBD) and the Nsp1 1-179 insert from existing clone DU66413 (pGEX6P1 2019-nCoV Nsp1). Nsp1 1-179-Intein CBDsuggested: NoneSoftware and Algorithms Sentences Resources (PepMap C18 100A - 300µm. PepMapsuggested: (BioWorks, RRID:SCR_014594)The MaxQuant output protein group text files were processed using Perseus software suite (Tyanova et al, 2016), version 1.6.10.45 was used. MaxQuantsuggested: (MaxQuant, RRID:SCR_014485)Perseussuggested: (Perseus, RRID:SCR_015753)The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al, 2019) partner repository with the dataset identifier PXD022904. PRIDEsuggested: (Pride-asap, RRID:SCR_012052)Mass spectra were acquired with a RapifleX MALDI-TOF/TOF instrument from Bruker Daltonics equipped with Compass software for FlexSeries 2.0. Compasssuggested: (COMPASS, RRID:SCR_015874)Data sets were further processed using Excel or GraphPad Prism (v7.03; Excelsuggested: NoneGraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPad Prism was utilized for graphical representation of the data. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.12.09.417741: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
About SciScore
SciScore is …
SciScore for 10.1101/2020.12.09.417741: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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