Endomembrane targeting of human OAS1 p46 augments antiviral activity

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1. 

   Version published to 10.7554/elife.71047 on eLife

   Aug 3, 2021
2. 

   ScreenIT

   Apr 24, 2021

   SciScore for 10.1101/2021.04.21.440697: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Both studies were approved by the Institutional Review Board at Benaroya Research Institute (IRB20-036 and IRB07109 respectively).
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibody (Table S3) incubation with antibodies against OAS1 (CST)	OAS1 suggested: None
   24h after transfection, cells were lysed in Co-IP (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP40; 1 mM EDTA) buffer and OAS1 proteins were immunoprecipitated from the lysate using 20 µg anti-FLAG antibody (Sigma) and Dynabeads Protein G.	anti-FLAG suggested: None
   Cells were …

   SciScore for 10.1101/2021.04.21.440697: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Both studies were approved by the Institutional Review Board at Benaroya Research Institute (IRB20-036 and IRB07109 respectively).
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibody (Table S3) incubation with antibodies against OAS1 (CST)	OAS1 suggested: None
   24h after transfection, cells were lysed in Co-IP (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP40; 1 mM EDTA) buffer and OAS1 proteins were immunoprecipitated from the lysate using 20 µg anti-FLAG antibody (Sigma) and Dynabeads Protein G.	anti-FLAG suggested: None
   Cells were washed three times with PBS and then stained with the secondary antibodies goat anti-rabbit IgG Alexa Fluor 488 (Themo Fisher) and goat anti-mouse IgG Alexa Fluor 648 (Thermo Fisher) in PBS containing 1% BSA and 0.3% Triton X-100 for 1 hour in the dark at room temperature (Table S2).	anti-mouse IgG suggested: (GenWay Biotech Inc. Cat# GWB-648F4B, RRID:AB_10276759)
   WNV infected cells were washed three times with PBS and then stained with the secondary antibodies goat anti-rabbit IgG Alexa Fluor 488 (Themo Fisher)	anti-rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T, A549, Vero, PH5CH8, Huh7, and HeLa cells were grown in DMEM (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin-streptomycin-glutamine (	HEK293T suggested: None A549 suggested: None HeLa suggested: None
   OAS1 and RNASEL KO 293T were generated using lentiviral transduction as described previously followed by selection in 2 μg/mL puromycin or blasticidin (Lau et al., 2015)	RNASEL KO 293T suggested: None
   G into 293FT cells for lentiviral packaging.	293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   293T cells were transduced with ACE2-expressing lentivirus and selected with puromycin (2 mg/mL) for 4 days to generate 293T-ACE2 cells.	293T suggested: RRID:CVCL_YZ65)
   THP-1 cells were differentiated with PMA for 24h.	THP-1 suggested: None
   For EMCV, WNV, CVB, and IAV infections, OAS1 KO 293T cells were seeded in 12 well plates coated with 10 μg/mL poly-L-ornithine hydrobromide (Sigma) and allowed to adhere overnight.	OAS1 KO 293T suggested: None
   For EMCV and WNV, titration was performed on Vero cells grown to 90% confluency in 6-well plates.	Vero suggested: None
   IAV was titered on MDCK cells grown to 90% confluency in 12-well plates.	MDCK suggested: None
   For infections, 293T-ACE2 cells seeded in 24-well plates (100,000 cells/well) were transfected with 250ng of plasmid (Empty vector, p42, p46, and p46 ATIL) using the TransIT X2 kit (Mirus).	293T-ACE2 suggested: RRID:CVCL_YZ65)
   Supernatants were harvested at 48hpi, and serial dilutions were tittered on Vero E6 cells seeded at 90% confluency in 12-well plates.	Vero E6 suggested: None
   RNA immunoprecipitation: OAS1 KO Huh7 cells were seeded the day before transfection with FLAG-p42, FLAG-p42CTIL, FLAG-p46, FLAG-p46ATIL or an EV control using TransIT-X2 (Mirus Bio).	Huh7 suggested: None
   Recombinant DNA
   Sentences	Resources
   Generation of knockout cell lines using CRISPR/Cas9 gene editing: Cloning of OAS1 targeting guide RNA (gRNA) 5′-GTGCATGCGGAAACACGTGTCTGG-3′ into pRRLU6-empty-gRNA-MND-Cas9-t2A-Puro vector or RNase L targeting gRNA 5′-GTTATCCTCGCAGCGATTGCGGGG-3′ into pRRLU6-empty-gRNA-MND-Cas9-t2A-Blast was achieved using the In-Fusion enzyme mix (Clontech).	pRRLU6-empty-gRNA-MND-Cas9-t2A-Puro suggested: None pRRLU6-empty-gRNA-MND-Cas9-t2A-Blast suggested: None
   This amplicon was cloned into a pLEX lentiviral backbone cut with BamHI and XhoI using the InFusion HD kit (Takara).	pLEX suggested: RRID:Addgene_117987)
   pLEX-ACE2 was co-transfected with psPAX2 and pMD2.	pLEX-ACE2 suggested: None psPAX2 suggested: RRID:Addgene_12260) pMD2 suggested: None
   N-terminal FLAG-tagged versions of OAS1 p42 and p46 were generated by cutting pcDNA3.1 OAS1 p42, p42CTIL, p46 and p46ATIL with BamHI and cloning of a 3xFLAG fragment by PCR amplification from pEF FLAG-ZAP-L (Schwerk et al., 2019) and Gibson assembly using primers 5′-CGACTCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGATGGACTACAAAGAC-3′ and 5′-GTCCAGAGATTTGGCTGGGGTATTTCTG AGATCCATCATGCTTGTCATCGTCATCCTTGTAATCGATG-3′ (Table S1).	pcDNA3.1 OAS1 suggested: None pEF FLAG-ZAP-L suggested: None
   Expression plasmids encoding p42DADA, p46DADA in pCDNA3.1 were generated by site-directed mutagenesis on pCDNA3.1 p42 or p46.	pCDNA3.1 suggested: RRID:Addgene_79663) pCDNA3.1 p42 suggested: None
   Briefly, pcDNA3.1 FLAG-OAS1 p46 was cut with KflI and ApaI. gBlocks were PCR-amplified using the following primer pair 5′-TAAGAATTGGGATGGGTCCCCAG-3′ and 5′-GACACTATAGAATAGGGCCCTCTAGA-3′ and then cut with with KflI and ApaI.	pcDNA3.1 FLAG-OAS1 p46 suggested: None
   293T OAS1 KO cells were incubated with 25 µM geranylgeranylalcohol azide (GGAA) and transfected with 250 ng/mL pCDNA3.1 FLAG-tagged OAS1 expression constructs 3h after addition of GGAA.	pCDNA3.1 FLAG-tagged suggested: None
   Software and Algorithms
   Sentences	Resources
   Gibson assembly compatible sequences for the unique portions for p46, p46ATIL, and p48, were generated by PCR amplification of gBlocks.	gBlocks suggested: (Gblocks, RRID:SCR_015945)
   Flow cytometry was performed on a BD FACS Canto II. % VSV-GFP+ cells were quantified using FlowJo (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Quantification of co-localization was performed using the Fiji Coloc 2 plugin.	Fiji suggested: (Fiji, RRID:SCR_002285)
   Tests for association between cases and controls were performed by logistic regression using PLINK, with sex, age (as of April 1, 2020), deprivation score of residential postal code, and the first 10 principal components as covariates.	PLINK suggested: (PLINK, RRID:SCR_001757)
   Statistics: Statistical analyses (other than genetic analysis) were performed with Prism 8 and the specific statistical analyses performed are indicated in the figure legends.	Prism suggested: (PRISM, RRID:SCR_005375)

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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: 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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3. 

   Version published to 10.1101/2021.04.21.440697v1 on bioRxiv

   Apr 22, 2021