A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor
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Abstract
The main protease, M pro , is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting M pro proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the M pro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with M pro in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in M pro completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of M pro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based M pro sensors revealed a molecular crowding-mediated increase in the rate of M pro activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 M pro and functional genomics application to determine the effect of sequence variation in M pro .
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SciScore for 10.1101/2022.01.31.478460: (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
Antibodies Sentences Resources Blots were incubated either with anti-His antibody (6x-His Tag Monoclonal Antibody (HIS.H8), Alexa Fluor 488; ThermoFisher Scientific-MA1-21315-A488; 1:5000) or with anti-Strep-tag mouse monoclonal antibody (anti-Strep-tag mouse monoclonal, C23.21; PROGEN-910STR; 1:5000) overnight at 4°C in dilution buffer (TBS-T containing 5% bovine serum albumin (BSA). anti-Hissuggested: Noneanti-Strep-tagsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, HEK 293T cells were seeded onto 96-well white plates before 24 h of transfection. HEK 293Tsuggested: NoneRecombinant DNA Sentences Resources For … SciScore for 10.1101/2022.01.31.478460: (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
Antibodies Sentences Resources Blots were incubated either with anti-His antibody (6x-His Tag Monoclonal Antibody (HIS.H8), Alexa Fluor 488; ThermoFisher Scientific-MA1-21315-A488; 1:5000) or with anti-Strep-tag mouse monoclonal antibody (anti-Strep-tag mouse monoclonal, C23.21; PROGEN-910STR; 1:5000) overnight at 4°C in dilution buffer (TBS-T containing 5% bovine serum albumin (BSA). anti-Hissuggested: Noneanti-Strep-tagsuggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, HEK 293T cells were seeded onto 96-well white plates before 24 h of transfection. HEK 293Tsuggested: NoneRecombinant DNA Sentences Resources For this, fragments BstXI-mNG-Mpro-Nter-auto-NLuc-XhoI and BstXI-mNG-Mpro-Nter-auto-L-NLuc-XhoI were synthesized (Integrated DNA Technologies, IDT; Iowa, USA) and inserted into pIDTSmart (Kan) vectors to generate the plasmid constructs pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc, respectively. pIDT-mNG-Mpro-Nter-auto-NLucsuggested: NonepIDT-mNG-Mpro-Nter-auto-L-NLucsuggested: NoneRestriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing. pmNeonGreen-DEVD-NLucsuggested: NoneFor bacterial expression and purification of the Mpro sensor, the mNG-Mpro-Nter-auto-NLuc plasmid construct was digested with HindIII and XhoI and the mNG-Mpro-Nter-auto-NLuc fragment was subcloned into similarly digested pET-28b(+) plasmid. mNG-Mpro-Nter-auto-NLucsuggested: NonepET-28b(+ )suggested: NoneLive cell, BRET-based Mpro proteolytic cleavage activity assays: Live cell Mpro proteolytic cleavage activity assays were performed by co-transfecting HEK 293T cells with either the pmNG-Mpro-Nter-auto-NLuc or the pmNG-Mpro-Nter-auto-L-NLuc Mpro sensor plasmid constructs along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) (a gift from Nevan Krogan (Addgene plasmid # 141370; http://n2t.net/addgene:141370 ; RRID:Addgene_141370)85or pLVX-EF1alpha-SARS-CoV-2-nsp5-C145A-2xStrep-IRES-Puro (C145A mutant Mpro) plasmid (a gift from Nevan Krogan (Addgene plasmid # 141371 ; http://n2t.net/addgene:141371 ; RRID:Addgene_141371)85 in 96-well white flat bottom plates (Nunc; 136101). pmNG-Mpro-Nter-auto-L-NLucsuggested: Nonedetected: RRID:Addgene_141370)detected: RRID:Addgene_141371)For dose-response experiments, the filler plasmid (a pcDNA3.1-based plasmid) is also co-transfected. pcDNA3.1-basedsuggested: NoneLive cell Mpro proteolytic cleavage inhibitor assay: HEK 293T cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc or pmNG-Mpro-Nter-auto-L-NLuc plasmid along with either pLVX-EF1alpha-SARS-CoV-2-nsp5-2xStrep-IRES-Puro (Mpro WT) ( pmNG-Mpro-Nter-auto-NLucsuggested: NoneLive cell, FlipGFP-based Mpro proteolytic assay: For live cell FlipGFP-based Mpro proteolytic activity assays, HEK 293T cells were seeded onto 24-well plates and co-transfected with the FlipGFP sensor plasmid (pcDNA3 FlipGFP(Mpro) pcDNA3suggested: RRID:Addgene_15475)Software and Algorithms Sentences Resources Mpro N-terminal autocleavage sequence analysis: A total of 1984 sequences for the SARS-CoV-2 pp1a polyprotein available at the NCBI Virus database (https://www.ncbi.nlm.nih.gov/genome/viruses/) were downloaded and aligned using MAFFT server (https://mafft.cbrc.jp/alignment/server/)63, 64. https://www.ncbi.nlm.nih.gov/genome/viruses/suggested: (NCBI Viral Genomes, RRID:SCR_013789)MAFFTsuggested: (MAFFT, RRID:SCR_011811)Models were generated using MODELLER (10.1 release, Mar. 18, 2021)66. MODELLERsuggested: (MODELLER, RRID:SCR_008395)The NAMD output structure was then used as an input for Gaussian accelerated molecular dynamics (GaMD) simulation utilizing the integrated GaMD module in NAMD and its default parameters73, 74 which included 2 ns of conventional molecular dynamics (cMD) equilibration run in GaMD, to collect potential statistics required for calculating the GaMD acceleration parameters, and another 50 ns equilibration run in GaMD after adding the boost potential74, 75, and finally GaMD production runs for 1000 ns. NAMDsuggested: (NAMD, RRID:SCR_014894)Restriction enzymes BstX-I and XhoI were used to excise the two DNA fragments of interests from entry clones pIDT-mNG-Mpro-Nter-auto-NLuc and pIDT-mNG-Mpro-Nter-auto-L-NLuc and ligated into similarly digested destination plasmid pmNeonGreen-DEVD-NLuc [Addgene: 98287]49 and further confirmed by Sanger sequencing. Addgenesuggested: (Addgene, RRID:SCR_002037)The ImageJ macro script used for the analysis is provided in the Supporting Text. ImageJsuggested: (ImageJ, RRID:SCR_003070)Data analysis and figure preparation: GraphPad Prism (version 9 for macOS, GraphPad Software, La Jolla California USA; www.graphpad.com), in combination with Microsoft Excel, was used for data analysis and graph preparation. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)Figures were assembled using Adobe Illustrator. Adobe Illustratorsuggested: (Adobe Illustrator, RRID:SCR_010279)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.
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.
Results from scite Reference Check: We found no unreliable references.
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