Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism
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Abstract
Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8 , the C-terminus with His 8 -Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.
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SciScore for 10.1101/2020.07.20.211623: (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
Antibodies Sentences Resources This anti-Flag antibody generate a prominent cross-reacting band at around 50 kDa which was used for loading normalization. anti-Flagsuggested: NoneFor proteins running at the same size of the cross-reacting band (Nsp15 and Nsp12), an anti-M2-FLAG antibody (Sigma-Aldrich, F1804) was used in combination with anti-GAPDH (BioRad, VPA00187) as the loading control. anti-M2-FLAGsuggested: Noneanti-GAPDHsuggested: NoneExperimental Models: Cell Lines Sentences Resourc… SciScore for 10.1101/2020.07.20.211623: (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
Antibodies Sentences Resources This anti-Flag antibody generate a prominent cross-reacting band at around 50 kDa which was used for loading normalization. anti-Flagsuggested: NoneFor proteins running at the same size of the cross-reacting band (Nsp15 and Nsp12), an anti-M2-FLAG antibody (Sigma-Aldrich, F1804) was used in combination with anti-GAPDH (BioRad, VPA00187) as the loading control. anti-M2-FLAGsuggested: Noneanti-GAPDHsuggested: NoneExperimental Models: Cell Lines Sentences Resources Mass spectrometry: HEK293 cells with stably integrated N-terminal tagged N protein (from plasmid 22) were induced with 1 μg/ml doxycyline for 0, 6, and 18 hours. HEK293suggested: NoneSoftware and Algorithms Sentences Resources The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD020339 (Perez-Riverol et al, 2019). PRIDEsuggested: (Pride-asap, RRID:SCR_012052)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 10.1101/2020.07.20.211623: (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 The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. HEK293suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045Data from additional tools added to each annotation on a weekly basis.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered …
SciScore for 10.1101/2020.07.20.211623: (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 The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. HEK293suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045Data from additional tools added to each annotation on a weekly basis.
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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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