SARS-CoV-2 hijacks p38β/MAPK11 to promote virus replication

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1. 

   Version published to 10.1101/2021.08.20.457146v2 on bioRxiv

   Sep 14, 2022
2. 

   ScreenIT

   Aug 23, 2021

   SciScore for 10.1101/2021.08.20.457146: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	Lysates were probe sonicated on ice 3 × 1s at 50% power, with 5s of rest in between pulses.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were incubated in primary antibody (1:1000 mouse anti-SARS N IC7 antibody, a kind gift from Thomas Moran) in antibody buffer (1% w/v BSA, 0.03% v/v Triton X-100, 0.1% fish gelatin in PBS) overnight at 4C.	anti-SARS N IC7 suggested: None
   Cells were incubated in 1:1000 anti-mouse AlexaFluor488 or anti-mouse AlexaFluor594 (Thermo Fisher Scientific) and 4’,6-diamidino-2-phenylindole counterstain (DAPI, Thermo …

   SciScore for 10.1101/2021.08.20.457146: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	Lysates were probe sonicated on ice 3 × 1s at 50% power, with 5s of rest in between pulses.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were incubated in primary antibody (1:1000 mouse anti-SARS N IC7 antibody, a kind gift from Thomas Moran) in antibody buffer (1% w/v BSA, 0.03% v/v Triton X-100, 0.1% fish gelatin in PBS) overnight at 4C.	anti-SARS N IC7 suggested: None
   Cells were incubated in 1:1000 anti-mouse AlexaFluor488 or anti-mouse AlexaFluor594 (Thermo Fisher Scientific) and 4’,6-diamidino-2-phenylindole counterstain (DAPI, Thermo Fisher Scientific) in antibody buffer at room temperature for one hour.	anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   CCL-185), HEK 293T cells, a human kidney epithelial cell line (HEK 293T/17, ATCC®, CRL-11268), and Vero E6 cells (Vero 76, clone E6, Vero E6, ATCC® CRL-1586), an African Green Monkey kidney epithelial cell line, were authenticated by ATCC.	HEK 293T suggested: ATCC Cat# CRL-11268G-1, RRID:CVCL_UE07) HEK 293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   A monoclonal ACE2-expressing A549 cell line was a kind gift from Brad Rosenberg.	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Virus stocks were grown on Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Cell viability assay: 2 × 104 ACE2-A549 cells in a 96-well white-bottom plate were transfected in triplicate with siRNA pools.	ACE2-A549 suggested: None
   Software and Algorithms
   Sentences	Resources
   For all analyses, samples were injected on a C18 reverse phase column (30 cm x 75 μm (ID)) packed with ReprosilPur 1.9 μm particles).	ReprosilPur suggested: None
   Protein abundance samples were fractioned with Field Assymetric Ion Mobility Spectrometry (FAIMS) fractionation with a FAIMS Pro device (Thermo Fisher Scientific).	Field Assymetric suggested: None
   Lysates were run on an SDS-PAGE gel with a protein ladder standard (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Plate was read for luminescence end-point kinetics with a 1s integration on a Cytation 5 Plate Reader using Gen5 software (Biotek Instruments)	Gen5 suggested: (Gen5, RRID:SCR_017317)
   GO-term enrichment was performed using Biojupies (Torre et al. 2018) and visualized using ggplot2 (Wickham 2016).	Biojupies suggested: (BioJupies, RRID:SCR_016346)
   Alignments to viral genomes was performed using bowtie2 (Langmead and Salzberg, 2012).	bowtie2 suggested: (Bowtie 2, RRID:SCR_016368)
   Genome coverage (viral gene counts at each nucleotide position) was analyzed using Integrative Genomics Viewer and visualized with ggplot2 (Robinson et al., 2011)	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
   Genome schematic was created with BioRender (BioRender.com)	BioRender suggested: (Biorender, RRID:SCR_018361)
   Spectronaut reports were analyzed by the MSstats package in the Rstudio statistical programming environment (Bruderer et al., 2017).	MSstats suggested: (MSstats, RRID:SCR_014353) Rstudio suggested: (RStudio, RRID:SCR_000432)
   Distances were calculated based on Euclidian distance using the dist function in R and the data were hierarchically ordered using the hclust function in R.	hclust suggested: (HCLUST, RRID:SCR_009154)

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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:

   One limitation of this approach is that it cannot determine whether the substrates identified were direct substrates of the kinase of interest. SARS-CoV-2 N contains p38-dependent phosphosites: Throughout the viral life cycle, coronavirus nucleocapsid protein (N) performs many crucial functions including oligomerizing along the length of viral RNA for protection, enhancing viral polymerase activity, modulating template switching, and innate immunity evasion (Chang et al., 2014). Post-translational modifications on N have been documented to affect N activities. Phosphorylation of avian Gammacoronavirus infectious bronchitis virus N has been shown to increase the affinity of N for viral RNA compared to non-viral RNA (Chen et al., 2005). Additionally, phosphorylation of Betacoronavirus murine hepatitis virus (MHV) N by GSK-3 has been shown to regulate replication of genomic RNA versus subgenomic RNA by promoting template read-through (Wu et al., 2014). In this study, we identified phosphosites on SARS-CoV-2 N that were p38-dependent, although their fold-changes were more modest compared to that of host p38 substrates that were quantified. As N phosphorylation is known to affect its activity, it is plausible that p38-dependent N phosphorylation is responsible for the phenotypes we observed, but we cannot exclude the possibility that p38-dependent phosphorylation of a host protein may drive these phenotypes. Further experiments are required to determine the impact of novel p38ß ph...

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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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.08.20.457146v1 on bioRxiv

   Aug 20, 2021