Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.09.11.291716: (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	Cell culture: Lenti-X 293T™ cells (H. sapiens; sex: female) obtained from Takara (cat. #632180) and Huh-7.5 cells (H. sapiens; sex: male) (Blight et al., 2002) were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Fisher Scientific, cat. #11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. #11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. #AUJ35777)
   Cell Line Authentication	Contamination: Both cell lines have tested negative for contamination with mycoplasma.

   Ta…

   SciScore for 10.1101/2020.09.11.291716: (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	Cell culture: Lenti-X 293T™ cells (H. sapiens; sex: female) obtained from Takara (cat. #632180) and Huh-7.5 cells (H. sapiens; sex: male) (Blight et al., 2002) were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Fisher Scientific, cat. #11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. #11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. #AUJ35777)
   Cell Line Authentication	Contamination: Both cell lines have tested negative for contamination with mycoplasma.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Viral titers were measured on Huh-7.5 cells by standard plaque assay.	Huh-7.5 suggested: RRID:CVCL_7927)
   To deliver the lentiGuidePurov2 sgRNA library, 3.6 × 107 Huh-7.5-Cas9 cells were transduced by spinoculation at 1,000 g × 1 h in media containing 4 μg/ml polybrene (Millipore, cat. #TR-1003-G) and 20 mM HEPES (Gibco, cat. #15630080) at a MOI = 0.14 to achieve ∼1,500-fold overrepresentation of each sgRNA.	Huh-7.5-Cas9 suggested: None
   The inocula of HCoV-229E, HCoV-NL63 and SARS-CoV-2 were supplemented with 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich, cat. #T1426) increasing the rate of infection.	HCoV-NL63 suggested: RRID:CVCL_RW88)
   Specifically, count tables for poly-A selected libraries from bulk RNA sequenced Calu-3 cells were used and processed with the same limma-Voom read normalization scheme above.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Software and Algorithms
   Sentences	Resources
   For scRNAseq analysis, Seurat objects were downloaded from FigShare (https://doi.org/10.6084/m9.figshare.12436517) (Chua et al., 2020).	FigShare suggested: (FigShare, RRID:SCR_004328)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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2. Version published to 10.1016/j.chom.2020.12.009

   Feb 1, 2021
3. 

   ScreenIT

   Sep 12, 2020

   SciScore for 10.1101/2020.09.11.291716: (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

   Cell culture Lenti-X 293T™ cells (H. sapiens; sex: female) obtained from Takara (cat. # 632180) and Huh-7.5 cells (H. sapiens; sex: male) (Blight et al., 2002) were maintained at 37 °C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Fisher Scientific, cat. # 11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. # 11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. # AUJ35777).

   Cell Line Authentication

   Both cell lines have tested negative for contamination with mycoplasma.

   Table 2: …

   SciScore for 10.1101/2020.09.11.291716: (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

   Cell culture Lenti-X 293T™ cells (H. sapiens; sex: female) obtained from Takara (cat. # 632180) and Huh-7.5 cells (H. sapiens; sex: male) (Blight et al., 2002) were maintained at 37 °C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Fisher Scientific, cat. # 11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. # 11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. # AUJ35777).

   Cell Line Authentication

   Both cell lines have tested negative for contamination with mycoplasma.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Lentivirus production To produce the lentiGuidePurov2 sgRNA lentivirus library, Lenti-X 293TTM cells were seeded in 25 ml DMEM supplemented with 0.1 mM NEAA and 3% FBS at 6.25 x 106 per dish in four poly-L-lysine coated p150 dishes.	293TTM suggested: None
   To generate working stocks, Huh-7.5 cells were infected at a multiplicity of infection (MOI) of 0.01 plaque forming unit (PFU)/cell (SARS-CoV-2,	Huh-7.5 suggested: RRID:CVCL_7927)
   CRISPR-Cas9 genetic screen Huh-7.5-Cas9 cells transduced with the lentiGuidePurov2 sgRNA library were seeded in p150 plates at 4.5 x 106 cells/plate in triplicate for each condition (mock, HCoV-229E, HCoV-NL63, HCoV-OC43, and SARS-CoV-2).	Huh-7.5-Cas9 suggested: None
   The inocula of HCoV-229E, HCoV-NL63 and SARS-CoV-2 were supplemented with 1 µg/ml TPCKtreated trypsin (Sigma-Aldrich, cat. # T1426) increasing the rate of infection.	HCoV-NL63 suggested: RRID:CVCL_RW88)
   For Calu-3 cells, data was obtained from GSE148729 (Wyler et al., 2020).	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
   Software and Algorithms
   Sentences	Resources
   For scRNAseq analysis, Seurat objects were downloaded from FigShare (https://doi.org/10.6084/m9.figshare.12436517) (Chua et al., 2020).	FigShare suggested: (FigShare, RRID:SCR_004328)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   LIMITATIONS While our study sheds new light on how host proteins are co-opted during coronavirus infection, it is not without limitations. As with all pooled CRISPR gene disruption screens, we expect that functionally redundant genes may not score (Ewen-Campen et al., 2017)). Additionally, the results of these genetic screens should not be interpreted to validate specific physical associations, as it is a purely functional readout. Additional biochemical studies would be necessary to test each predicted interaction. Nevertheless, we identified robust pan-coronavirus and discrete strain-specific host factors that scored with up to 10 sgRNAs scoring per gene, validating the utility of this near-saturation CRISPR-based genetic screening approach. We acknowledge that Huh-7.5 cells, which were chosen based on their unique capacity to support infection by multiple coronaviruses and also because they constitutively express 324/332 of the SARS-CoV-2 interactome factors, are different than the HEK293T system used by Gordon et al. to generate the PPI network probed in this study and are not airway cells. However, this robust and experimentally tractable system offers a unique advantage to directly compare hard-wired host factor requirements across multiple viruses in a cell-type- and tissue-type-agnostic manner, which can then be validated using gold-standard primary cells or animal models. Future studies will be required to dissect the specific requirement for each member of the PPI n...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   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.

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   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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4. 

   Version published to 10.1101/2020.09.11.291716v1 on bioRxiv

   Sep 11, 2020