Comparative Multiplexed Interactomics of SARS-CoV-2 and Homologous Coronavirus Nonstructural Proteins Identifies Unique and Shared Host-Cell Dependencies

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1. Version published to 10.1021/acsinfecdis.0c00500

   Dec 2, 2020
2. 

   ScreenIT

   Jul 15, 2020

   SciScore for 10.1101/2020.07.13.201517: (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
   After blocking, cells were incubated with anti-PDIA4 primary antibody (Protein Tech, 14712-1-AP) in blocking buffer (1:1000 dilution) for 1 hour at 37°C.	anti-PDIA4 suggested: None
   After three PBS washes, cells were incubated with AlexFluor 488-conjugated anti-rabbit goat antibody (ThermoFisher, A-11008) in blocking buffer (1:500 dilution) at room temperature for 30 min.	anti-rabbit goat suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)
   Cells were then stained with M2 FLAG primary antibody (SigmaAldrich, F1804) and AlexFluor 594-conjugated anti-mouse goat antibody (ThermoFisher, A-11005) using the …

   SciScore for 10.1101/2020.07.13.201517: (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
   After blocking, cells were incubated with anti-PDIA4 primary antibody (Protein Tech, 14712-1-AP) in blocking buffer (1:1000 dilution) for 1 hour at 37°C.	anti-PDIA4 suggested: None
   After three PBS washes, cells were incubated with AlexFluor 488-conjugated anti-rabbit goat antibody (ThermoFisher, A-11008) in blocking buffer (1:500 dilution) at room temperature for 30 min.	anti-rabbit goat suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)
   Cells were then stained with M2 FLAG primary antibody (SigmaAldrich, F1804) and AlexFluor 594-conjugated anti-mouse goat antibody (ThermoFisher, A-11005) using the same conditions.	anti-mouse goat suggested: (Molecular Probes Cat# A-11005, RRID:AB_141372)
   Experimental Models: Cell Lines
   Sentences	Resources
   Immunofluorescence Confocal Microscopy: HEK293T cells were cultured on glass-bottom culture dishes (MatTek, P35G-0-14-C) and transfected with CoV expression constructs as previously described.	HEK293T suggested: None
   Software and Algorithms
   Sentences	Resources
   Peptide IDs were filtered using the Percolator node using an FDR target of 0.01.	Percolator suggested: (OMSSAPercolator, RRID:SCR_000287)
   Gene set enrichment analysis: GO-term categories for biological processes and cellular components for interactors were based on assignment in the Proteome Discoverer Protein Annotation node.	Gene set enrichment analysis suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199) Proteome Discoverer suggested: (Proteome Discoverer, RRID:SCR_014477)
   Finally, we searched the overlapping secondary interactors against the STRING database human proteome to determine interactors between secondary interactors with a threshold of greater than 50% likelihood in the experimental score category.	STRING suggested: (STRING, RRID:SCR_005223)
   The results were plotted in Cytoscape.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
   Cells were imaged using an LSM-880 confocal microscope (Zeiss) and images were merged using Image J software.	Image J suggested: (ImageJ, RRID:SCR_003070)

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   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).

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

   We acknowledge the limitations of using transiently transfected viral proteins for AP-MS. Viral infection results in a collection of both protein-protein and RNA-protein interactions and our approach cannot account for how these events influence the interactions of nsp2 and nsp4. However, given the logistical barriers to handling BSL-3 viruses, paired with the urgency of the current pandemic, our workflow is an efficient system to perform comparative analysis and generate a shortlist of interactors to prioritize for further investigation. We identify several nsp2 interactors shared across SARS strains, including STOML2, and prohibitins (PHB and PHB2), which were previously identified as interacting with SARS-CoV-117. These proteins work in tandem to induce formation of metabolically active mitochondrial membranes to regulate mitochondrial biogenesis. Increased levels of STOML2 are associated with increased ATP production and reduced apoptosis induction26. This conserved interaction for SARS strains presents an avenue for nsp2 to increase mitochondrial metabolism and stall apoptosis to maintain a pro-viral cellular environment. Additionally, STOML2 has been found to play a key role in stabilizing hepatitis C virus replication complexes55 and PHB has been shown to promote entry of both Chikungunya virus56 and enterovirus 7157. These factors may prove effective pan-RNA virus targets for host-directed therapies. For nsp4, we identify multiple unique SARS-CoV-1 interactors, most 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.

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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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3. 

   Version published to 10.1101/2020.07.13.201517v1 on bioRxiv

   Jul 15, 2020