Endomembrane targeting of human OAS1 p46 augments antiviral activity
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Abstract
Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.
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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) OAS1suggested: None24h 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-FLAGsuggested: NoneCells 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) OAS1suggested: None24h 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-FLAGsuggested: NoneCells 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 IgGsuggested: (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 IgGsuggested: NoneExperimental 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 ( HEK293Tsuggested: NoneA549suggested: NoneHeLasuggested: NoneOAS1 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 293Tsuggested: NoneG into 293FT cells for lentiviral packaging. 293FTsuggested: 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. 293Tsuggested: RRID:CVCL_YZ65)THP-1 cells were differentiated with PMA for 24h. THP-1suggested: NoneFor 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 293Tsuggested: NoneFor EMCV and WNV, titration was performed on Vero cells grown to 90% confluency in 6-well plates. Verosuggested: NoneIAV was titered on MDCK cells grown to 90% confluency in 12-well plates. MDCKsuggested: NoneFor 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-ACE2suggested: 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 E6suggested: NoneRNA 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). Huh7suggested: NoneRecombinant 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-Purosuggested: NonepRRLU6-empty-gRNA-MND-Cas9-t2A-Blastsuggested: NoneThis amplicon was cloned into a pLEX lentiviral backbone cut with BamHI and XhoI using the InFusion HD kit (Takara). pLEXsuggested: RRID:Addgene_117987)pLEX-ACE2 was co-transfected with psPAX2 and pMD2. pLEX-ACE2suggested: NonepsPAX2suggested: RRID:Addgene_12260)pMD2suggested: NoneN-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 OAS1suggested: NonepEF FLAG-ZAP-Lsuggested: NoneExpression plasmids encoding p42DADA, p46DADA in pCDNA3.1 were generated by site-directed mutagenesis on pCDNA3.1 p42 or p46. pCDNA3.1suggested: RRID:Addgene_79663)pCDNA3.1 p42suggested: NoneBriefly, 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 p46suggested: None293T 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-taggedsuggested: NoneSoftware and Algorithms Sentences Resources Gibson assembly compatible sequences for the unique portions for p46, p46ATIL, and p48, were generated by PCR amplification of gBlocks. gBlockssuggested: (Gblocks, RRID:SCR_015945)Flow cytometry was performed on a BD FACS Canto II. % VSV-GFP+ cells were quantified using FlowJo (Tree Star). FlowJosuggested: (FlowJo, RRID:SCR_008520)Quantification of co-localization was performed using the Fiji Coloc 2 plugin. Fijisuggested: (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. PLINKsuggested: (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. Prismsuggested: (PRISM, RRID:SCR_005375)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).
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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