Abstract The subset of patients who develop critical illness in Covid-19 have extensive inflammation affecting the lungs 1 and are strikingly different from other patients: immunosuppressive therapy benefits critically-ill patients, but may harm some non-critical cases. 2 Since susceptibility to life-threatening infections and immune-mediated diseases are both strongly heritable traits, we reasoned that host genetic variation may identify mechanistic targets for therapeutic development in Covid-19. 3 GenOMICC (Genetics Of Mortality In Critical Care, genomicc.org ) is a global collaborative study to understand the genetic basis of critical illness. Here we report the results of a genome-wide association study (GWAS) in 2244 critically-ill Covid-19 patients from 208 UK intensive care units (ICUs), representing >95% of all ICU beds. Ancestry-matched controls were drawn from the UK Biobank population study and results were confirmed in GWAS comparisons with two other population control groups: the 100,000 genomes project and Generation Scotland. We identify and replicate three novel genome-wide significant associations, at chr19p13.3 (rs2109069, p = 3.98 × 10 −12 ), within the gene encoding dipeptidyl peptidase 9 ( DPP9 ), at chr12q24.13 (rs10735079, p =1.65 × 10 −8 ) in a gene cluster encoding antiviral restriction enzyme activators ( OAS1, OAS2, OAS3 ), and at chr21q22.1 (rs2236757, p = 4.99 × 10 −8 ) in the interferon receptor gene IFNAR2 . Consistent with our focus on extreme disease in younger patients with less comorbidity, we detect a stronger signal at the known 3p21.31 locus than previous studies (rs73064425, p = 4.77 × 10 −30 ). We identify potential targets for repurposing of licensed medications. Using Mendelian randomisation we found evidence in support of a causal link from low expression of IFNAR2 , and high expression of TYK2 , to life-threatening disease. Transcriptome-wide association in lung tissue revealed that high expression of the monocyte/macrophage chemotactic receptor CCR2 is associated with severe Covid-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms, and mediators of inflammatory organ damage in Covid-19. Both mechanisms may be amenable to targeted treatment with existing drugs. Large-scale randomised clinical trials will be essential before any change to clinical practice.
Abstract The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole exome sequencing data of about 4,000 SARS-CoV-2-positive individuals were used to define an interpretable machine learning model for predicting COVID-19 severity. Firstly, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthly, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.
Severe COVID-19 is characterised by immunopathology and epithelial injury. Proteomic studies have identified circulating proteins that are biomarkers of severe COVID-19, but cannot distinguish correlation from causation. To address this, we performed Mendelian randomisation (MR) to identify proteins that mediate severe COVID-19. Using protein quantitative trait loci (pQTL) data from the SCALLOP consortium, involving meta-analysis of up to 26,494 individuals, and COVID-19 genome-wide association data from the Host Genetics Initiative, we performed MR for 157 COVID-19 severity protein biomarkers. We identified significant MR results for five proteins: FAS, TNFRSF10A, CCL2, EPHB4 and LGALS9. Further evaluation of these candidates using sensitivity analyses and colocalization testing provided strong evidence to implicate the apoptosis-associated cytokine receptor FAS as a causal mediator of severe COVID-19. This effect was specific to severe disease. Using RNA-seq data from 4,778 individuals, we demonstrate that the pQTL at the FAS locus results from genetically influenced alternate splicing causing skipping of exon 6. We show that the risk allele for very severe COVID-19 increases the proportion of transcripts lacking exon 6, and thereby increases soluble FAS. Soluble FAS acts as a decoy receptor for FAS-ligand, inhibiting apoptosis induced through membrane-bound FAS. In summary, we demonstrate a novel genetic mechanism that contributes to risk of severe of COVID-19, highlighting a pathway that may be a promising therapeutic target.
Abstract Pulmonary inflammation drives critical illness in Covid-19, 1;2 creating a clinically homogeneous extreme phenotype, which we have previously shown to be highly efficient for discovery of genetic associations. 3;4 Despite the advanced stage of illness, we have found that immunomodulatory therapies have strong beneficial effects in this group. 1;5 Further genetic discoveries may identify additional therapeutic targets to modulate severe disease. 6 In this new data release from the GenOMICC (Genetics Of Mortality in Critical Care) study we include new microarray genotyping data from additional critically-ill cases in the UK and Brazil, together with cohorts of severe Covid-19 from the ISARIC4C 7 and SCOURGE 8 studies, and meta-analysis with previously-reported data. We find an additional 14 new genetic associations. Many are in potentially druggable targets, in inflammatory signalling (JAK1, PDE4A), monocyte-macrophage differentiation (CSF2), immunometabolism (SLC2A5, AK5), and host factors required for viral entry and replication (TMPRSS2, RAB2A). As with our previous work, these results provide tractable therapeutic targets for modulation of harmful host-mediated inflammation in Covid-19.
Additional file 6: Table S5. Significantly enriched Gene Ontology terms for genes with variable and consistent expression across tissues in humans and cattle.
Abstract We have extensively mapped the genes responsible for hair colour in the UK population. MC1R mutations are well established as the principal genetic cause of red hair colour, but with variable penetrance. We find variation at genes encoding its agonist ( POMC ), inverse agonist ( ASIP ) and other loci contribute to red hair and demonstrate epistasis between MC1R and some of these loci. Blonde hair is associated with over 200 loci, and we find a genetic continuum from black through dark and light brown to blonde. Many of the associated genes are involved in hair growth or texture, emphasising the cellular connections between keratinocytes and melanocytes in the determination of hair colour.
Summary Cell identity is governed by gene expression, regulated by Transcription Factor (TF) binding at cis-regulatory modules. We developed the NetNC software to decode the relationship between TF binding and the regulation of cognate target genes in cell decision-making; demonstrated on nine datasets for the Snail and Twist TFs, and also modENCODE ‘HOT’ regions. Results illuminated conserved molecular networks controlling development and disease, with implications for precision medicine. Predicted ‘neutral’ TF binding accounted for the majority (50% to ≥80%) of candidate target genes from statistically significant peaks and HOT regions had high functional coherence. Expression of orthologous functional TF targets discriminated breast cancer molecular subtypes and predicted novel tumour biology. We identified new gene functions and network modules including crosstalk with notch signalling and regulation of chromatin organisation, evidencing networks that reshape Waddington’s landscape during epithelial remodelling. Predicted invasion role s were validated using a tractable cell model, supporting our computational approach.
Article Figures and data Abstract Introduction Materials and methods Results Discussion Data availability References Decision letter Author response Article and author information Metrics 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. Introduction Oligoadenylate synthetase (OAS) proteins are a family of interferon (IFN)-induced sensors of viral RNA critical for cell-intrinsic innate immune defense against viruses through activation of the latent endoribonuclease RNase L (Hornung et al., 2014). Recognition of viral double-stranded RNA (dsRNA) induces a conformational change in OAS proteins to reveal a catalytic pocket which converts ATP to the second messenger 2′−5′A (Lohöfener et al., 2015). Binding to 2′−5′A dimerizes and activates RNase L, which cleaves cellular and viral RNA in order to block viral replication (Han et al., 2014). Although the importance of RNase L in restricting a variety of viruses is well documented, it is unclear how individual OAS proteins contribute to this breadth of antiviral activity (Silverman, 2007). Interestingly, the C-terminal region of human OAS1 is alternatively spliced to produce the protein isoforms p42, p44, p46, p48, and p52, named according to their molecular weight (Bonnevie-Nielsen et al., 2005). All human OAS1 isoforms share the first five exons of OAS1, which contain the RNA binding and catalytic domains, but each isoform splices a distinct sixth exon to generate unique C-termini. The specific antiviral roles of individual human OAS1 isoforms are not defined. The production of OAS1 p46 is controlled by an SNP in the splice-acceptor site of exon six in OAS1 (rs10774671 A>G) (Figure 1A). This splice site SNP in OAS1 is associated with genetic susceptibility to multiple flaviviruses and autoimmune disorders (El Awady et al., 2011; Haralambieva et al., 2011; Liu et al., 2017; Simon-Loriere et al., 2015). The G allele of this SNP shifts the splice acceptor site in exon 6 by one nucleotide to generate p46, while other OAS1 isoforms, primarily p42, are produced when the A allele is present (Lim et al., 2009). OAS1 p46 is unique among OAS1 isoforms because it is the only isoform with a C-terminal CaaX (cysteine-aliphatic-aliphatic-any residue) motif. Proteins containing CaaX motifs at their C-termini undergo a post translational lipidation modification termed prenylation and are targeted to the cytosolic face of intracellular organelle membranes of the endomembrane system following post-prenylation processing at the endoplasmic reticulum (Wang and Casey, 2016). The significance of the CaaX motif in OAS1 p46 and whether endomembrane targeting might alter the antiviral activity of OAS1 is unknown. Figure 1 with 2 supplements see all Download asset Open asset The p46 isoform of OAS1 is targeted to the endomembrane system. (A) Differential C-terminal splicing of OAS1 creates isoform diversity. (B) Immunoblot analysis of OAS1 isoform expression across cell lines treated with 1000 U/mL rIFNβ for 24 hr (n=3). (C) Immunoblot analysis of OAS1 isoform expression in PBMCs from donors with indicated genotype at rs10774671 treated with 1000 U/mL rIFNβ for 24 hr. Ectopic expression of OAS1 p42 and p46 in OAS1 KO 293 T cells serves as control (three independent donors of each genotype depicted). (D) Immunoblot of OAS1 KO 293T whole cell lysate (left) and immunoprecipitated (right) FLAG-tagged p42, p46, p42CTIL, or p46ATIL constructs subjected to Click-chemistry reaction with geranylgeranyl azide and alkyne biotin; representative immunoblot of two independent experiments is shown. (E) Representative maximum intensity projections of the indicated cell lines treated with 1000 U/mL with rIFNβ for 24 hr followed by staining with anti-OAS1 antibody (green), anti-Golgin-97 (magenta), and DAPI (blue); representative cells from one out of three independently performed experiments are depicted. (F) Pearson’s correlation of OAS1 and Golgin-97 in individual cells from the indicated cell lines; each data point represents an individual cell from one representative experiment. (G) Representative confocal micrographs of OAS1 KO Huh7 transfected with constructs encoding p42, p46, p42CTIL, or p46ATIL stained with anti-OAS1 (green) and Golgin-97 (magenta) antibodies and DAPI (blue); (n=2). (H) Pearson’s correlation of OAS1 and Golgin-97 in OAS1 KO Huh7 cells expressing p42, p46, p42CTIL, or p46ATIL; each data point represents an individual cell of one representative experiment. Scale on micrographs in (E) and (G) = 5 μm. (F, H) Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test where **p<0.01, ****p<0001. Figure 1—source data 1 Uncropped gels for the associated panels in Figure 1 and Figure 1—figure suppleent 2. https://cdn.elifesciences.org/articles/71047/elife-71047-fig1-data1-v2.zip Download elife-71047-fig1-data1-v2.zip How the subcellular targeting of OAS family members impacts their specificity and antiviral activity is unclear. Most intracellular viral RNA (vRNA) sensors localize to the cytosol where they are poised to sense accumulating vRNA during infection (Ablasser and Hur, 2020; Chan and Gack, 2016). However, since many RNA viruses replicate their RNA in close association with intracellular membranes, placing OAS proteins at membranous compartments may augment vRNA sensing in certain contexts. Notably, positive-strand RNA viruses, such as flaviviruses, picornaviruses, and coronaviruses replicate their RNA within modified host organelles of the endomembrane system (Romero-Brey and Bartenschlager, 2014). These replication organelles shield vRNA from detection by cytosolic RNA sensors such as RIG-I (Neufeldt et al., 2016). Whether or not the host has evolved strategies to survey specific intracellular membranes for viral replication is unclear. We therefore hypothesized OAS1 p46, through its CaaX motif, is targeted to the endomembrane system and this targeting gives it enhanced access to vRNA during infection. In support of this model, we show the prenylated isoform, OAS1 p46, is targeted to Golgi membranes and this membrane-targeting results in enhanced detection of vRNA and augmented antiviral activity against positive-strand RNA viruses such as flaviviruses, picornaviruses, and SARS-CoV-2. Our human genetic data further supports the contribution of OAS1 rs10774671 to severity of COVID-19. More broadly, this work reveals how intracellular membrane targeting of OAS1 is critical for detecting human pathogenic viruses that replicate on organellar membranes. Materials and methods Cells, cell culture conditions, and treatments Request a detailed protocol All cells (Supplementary file 1) were incubated at 37°C with 5% CO2. HEK293T, A549, Vero, PH5CH8, Huh7, HeLa, and primary human fibroblasts were grown in DMEM (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin-streptomycin-glutamine (Mediatech). Daudi cells and PBMCs were cultured in RPMI 1640 (Sigma) containing 10% heat-inactivated FBS (Atlanta Biologicals) and 1% penicillin-streptomycin-glutamine (Mediatech). THP-1 cells were cultured in RPMI 1640 (Sigma) containing 10% heat-inactivated FBS (Atlanta Biologicals), 1% penicillin-streptomycin-glutamine (Mediatech), 10 mM Hepes (Corning), 1x NEAA (Corning), 1 mM sodium pyruvate (Corning), and 50 μM 2-mercaptoethanol (Sigma). Where applicable, THP-1 cells were differentiated in THP-1 media containing 40 nM of PMA (Sigma-Aldrich) for 24 hr. Recombinant human rIFNβ (PBL Interferon Source) was used at 200–1000 IU/mL. Primary human fibroblast lines were isolated from minced skin punch biopsies and subcultured to separate from epithelial and other cell types while grown in RPMI 1640 with 10% fetal calf serum supplemented with penicillin-streptomycin and gentamicin. Subculturing was performed with serial trypsinization and passage. Samples were consented under Seattle Children’s Institutional Review Board (protocol #11738). Cryopreserved PBMCs from healthy control subjects genotyped for OAS1 rs10774671 were obtained from the Benaroya Research Institute Immune Mediated Registry and Repository. All subjects provided informed consent and the study was approved by the Institutional Review Board (IRB07109). PBMC were isolated from heparinized peripheral blood by Ficoll-Hypaque centrifugation. All cell lines used were tested negative for mycoplasma and none of the cell lines belong to the group of commonly misidentified cell lines according to the ICLAC register. Cell lines were authenticated by microscopy and through ligand/PAMP specificity. Generation of knockout cell lines using CRISPR/Cas9 gene editing Request a detailed protocol 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). 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). Transient transfection was utilized to knockout OAS1 in A549 and Huh7 cells. Cells were transfected with OAS1 gRNA or a Cas9-expressing control vector using TransIT-X2 (Mirus Bio) according to the manufacturer’s instructions. At 24 hr post transfection, cells were selected with 2 μg/mL puromycin. Knockouts were validated by western blotting. Generation of 293T-ACE2 cells Request a detailed protocol Lentiviral expression vector for ACE2 (pLEX-ACE2) was generated by amplifying the ACE2 sequence from cDNA from Huh7 cells (5′- GACTCTACTAGAGGATCCGCCACCATGTCAAGCTCTTCCTGGCTCC-3′ and 5′- GGGCCCTCTAGACTCGAGCTAAAAGGAGGTCTGAACATCATCAGTG-3′). This amplicon was cloned into a pLEX lentiviral backbone cut with BamHI and XhoI using the InFusion HD kit (Takara). pLEX-ACE2 was co-transfected with psPAX2 and pMD2.G into 293FT cells for lentiviral packaging. 293 T cells were transduced with ACE2-expressing lentivirus and selected with puromycin (2 mg/mL) for 4 days to generate 293T-ACE2 cells. ACE2 expression was verified by immunoblot (Proteintech). Immunoblotting Request a detailed protocol Cells were lysed in RIPA buffer (+1× HALT protease and phosphatase inhibitor), and 10–30 μg total protein from whole-cell lysates was run on SDS–PAGE and transferred to polyvinylidene difluoride membranes (Thermo Scientific). The membranes were blocked in 5% milk in PBS-T (pPBS/Tween 20). Primary antibody (Supplementary file 2) incubation with antibodies against OAS1 (CST), RNase L (CST), or FLAG (Sigma) were performed in 5% milk in PBS-T overnight at 4°C. Membranes were washed for 5 min in PBS-T three times. Secondary antibody incubation was performed in 5% milk in PBS-T at room temperature for 1 hr and after membranes were washed for 5 min in PBS-T three times. Membranes were imaged on a Chemidoc XR system. OAS1 siRNA knockdown Request a detailed protocol Dicer-substrate short interfering RNAs against a common region of OAS1 or the unique 3′UTR of p42 or p46 were custom designed and procured from Integrated DNA Technologies (Supplementary file 3). THP-1 cells were differentiated with PMA for 24 hr. At 24 hr post treatment, cells were transfected with 20 nM of siRNA using TransIT-X2 (Mirus Bio) according to the manufacturer’s instructions. Viral infections were performed at 24 hr post-transfection. Cloning Request a detailed protocol Expression plasmids encoding OAS1 p42, p42CTIL, p44, p46, p46ATIL, p48, and p52 were generated by Gibson assembly of the common OAS1 sequence with the isoform-specific sequence into the pCDNA3.1 vector (Supplementary file 3). Empty pcDNA3.1 was cut using BamHI and XbaI. A Gibson assembly compatible fragment for the common sequence of OAS1 was PCR-amplified from an OAS1 expression plasmid (gift from Dan Stetson) using primers 5′-TGGTACCGAGCTCGATGATGGATCTCAGAA-3′ and 5′-CAGCAGAATCCAGG AGCTCACTGG-3′. Gibson assembly compatible fragments for the unique sequences of p42, p42CTIL, and p44 were generated by PCR amplification of annealed sense and antisense oligos (Supplementary file 3). Gibson assembly compatible sequences for the unique portions for p46, p46ATIL, and p48, were generated by PCR amplification of gBlocks. 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′ (Supplementary file 3). (Schwerk et al., 2019) and Gibson assembly using primers 5′-CGACTCACTATAGGGAGACCCA AGCTTGGTACCGAGCTCGATGGACTACAAAGAC-3′ and 5′--GTCCAGAGATTTGGCTGGGGTATTTCTGAGATCCATCATGCTTGTCATCGT CATCCTTGTAATCGATG-3′ (Supplementary file 3). Expression plasmids encoding p42DADA, p46DADA in pCDNA3.1 were generated by site-directed mutagenesis on pCDNA3.1 p42 or p46. FLAG-tagged expression plasmids encoding p46 common+CTIL, p46 alanine mutant 9, p46 D12aa, p46 D22aa, p46 D32aa, p42 CFK mutant, and p46 CFK mutant were generated by site-directed mutagenesis on pCDNA3.1 FLAG-p46. Site-directed mutagenesis was performed using the QuikChange Lightning kit (Agilent) according to the manufacturer’s instructions. All primers used for site-directed mutagenesis are listed in Supplementary file 3. OAS1 p46 C-terminal alanine mutants 1–8 and OAS1 p46 C-terminus species hybrids were generated by ligation of mutant gBlocks (Supplementary file 3). 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. Ligation was performed using T4 DNA ligase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Geranylgeranyl click chemistry immunoprecipitation Request a detailed protocol Geranylgeranyl click chemistry IP reactions were performed using the Click-iT labeling kit and reagents (Thermo Fisher) according to the manufacturer’s instructions with the following modifications. 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 3 hr after addition of GGAA. 24 hr 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. After five washes in Co-IP buffer, the Dynabeads were resuspended in 50 μl 50 mM Tris-HCl, pH 8, and the click chemistry reaction was performed according to the manufacturer’s instruction. Immunoprecipitated protein were immunoblotted and probed for presence of geranylgeranyl azide-biotin labeling using an HRP-conjugated streptavidin antibody. Virus infections and titer quantification Request a detailed protocol Virus and their sources are listed in Supplementary file 4. Encephalomyocarditis virus was grown and titered in Vero cells. West Nile virus Texas from Gale laboratory was grown as previously described (Aarreberg et al., 2019). CVB3-Nancy was prepared as previously described (Laufman et al., 2019). Influenza virus A/PR/8/34 and Influenza A virus A/Udorn/72 H3N2 R38A was prepared as described previously (Min and Krug, 2006). For EMCV, WNV, CVB, and IAV infections, OAS1 KO 293 T cells were seeded in 12 well plates coated with 10 μg/mL poly-L-ornithine hydrobromide (Sigma) and allowed to adhere overnight. Plasmids were then transfected using TransIT-X2 (Mirus Bio). At 24 hr post transfection, cells were infected with EMCV, WNV, or CVB at the indicated MOIs for 1 hr with gentle rocking. After 1 hr the inoculum was removed, and fresh media was added. For virus titer quantification, culture supernatants were serially diluted in DMEM using 96 well plates. For EMCV and WNV, titration was performed on Vero cells grown to 90% confluency in six-well plates. For CVB, titration was performed on HeLa cells grown to 90% confluency in six-well plates. IAV was titered on MDCK cells grown to 90% confluency in 12-well plates. The inoculum was removed after 1 hr of gentle rocking and a 0.8% agarose overlay was added containing the following: 0.8% UltraPure Low Melting Point Agarose (Thermo Fisher), 1x DMEM, 0.15% Sodium Bicarbonate, 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin-streptomycin-glutamine (Mediatech). For EMCV plates were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) at 24 hr post-infection followed by staining with a 5% crystal violet solution prepared by dissolving crystal violet (Sigma Aldrich) in a 50/50 mixture of 100% ethanol and deionized water. For IAV plates were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) at 24 hr post-infection followed by staining with a 5% crystal violet solution. For CVB plates were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) at 24 hr post-infection followed by staining with a 5% crystal violet solution. For WNV a neutral red overlay containing 0.01% neutral red (Sigma-Aldrich) 0.8% UltraPure Low Melting Point Agarose (Thermo Fisher), 1x DMEM, 0.15% sodium bicarbonate, 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals) and 1% penicillin-streptomycin-glutamine (Mediatech) was added at 48 hr post-infection and plaques were read 24 hr later. For quantification of Indiana vesiculovirus (VSV-GFP) replication, OAS1 KO 293 T cells transfected with OAS1 p42, p46 and EV for 24 hr prior to infection, were infected with Indiana vesiculovirus (VSV-GFP; MOI=0.5, 6 hr). Cells were harvested using trypsin, washed 2x with PBS, and incubated with Zombie NIR fixable viability dye (1:1000) (BioLegend) for 30 min at room temperature in PBS. Cells were washed 2x with PBS, fixed in 4% PFA for 10 min at room temperature, and then washed once with FACS buffer (PBS with 0.1%BSA). Flow cytometry was performed on a BD FACS Canto II. % VSV-GFP+ cells were quantified using FlowJo (Tree Star). SARS-CoV-2 strain USA/WA-1/2020 was propagated and titered on VeroE6 cells (gift of Dr. Ralph Baric). For infections, 293T-ACE2 cells seeded in 24-well plates (100,000 cells/well) were transfected with 250 ng of plasmid (Empty vector, p42, p46, and p46 ATIL) using the TransIT X2 kit (Mirus). A duplicate plate was transfected at the same time in order to confirm OAS1 expression by immunoblot. 24 hr post-transfection, cells were infected with SARS-CoV-2 at an MOI of 0.1 in serum-free DMEM for 1 hr, and media was replenished with DMEM containing 4% serum. Supernatants were harvested at 48hpi, and serial dilutions were tittered on Vero E6 cells seeded at 90% confluency in 12-well plates. Inoculum was removed after 1 hr of gentle rocking and replenished with an agarose overlay containing 0.4% Noble Agar (Thermo Fisher) in DMEM containing 10% serum. At 72 hpi, plates were fixed with 10% formaldehyde and stained with crystal violet solution (0.1% crystal violet and 20% methanol in water). Confocal laser scanning microscopy Request a detailed protocol For all microscopy experiments, cells were seeded on #1.5 12 mm coverslips (Bioscience Tools) coated with 10 μg/mL poly-L-ornithine hydrobromide (Sigma) and allowed to adhere overnight. All antibodies used in this study are listed in Supplementary file 2. For experiments testing endogenous OAS1 localization, cells were treated with rIFNβ for 24 hr. For experiments testing OAS1 localization in dox-inducible OAS1 A549 cells, cells were treated with 200 ng/mL doxycycline for 24 hr. For experiments testing localization of OAS1 in Huh7 cells, plasmids were transfected for 24 hr using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s instructions. At 24 hr post treatment/expression, cells were washed with PBS and then fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 10 min at room temperature, washed with PBS, and then permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature. Cells were washed with PBS and then resuspended in a 3% BSA/PBS blocking solution for 1 hr. After blocking, cells were stained with rabbit anti-OAS1 (CST) and mouse IgG1 anti-Golgin 97 in PBS containing 1% BSA and 0.3% Triton X-100 for 1 hr in the dark at room temperature. 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 hr in the dark at room temperature. Samples were washed once with PBS, stained with DAPI in PBS for 10 min in the dark, followed by washing three times with PBS and then mounted with ProLong Glass antifade mounting media (Thermo Fisher). Samples were cured in the dark at room temperature for 24–48 hr prior to imaging. For experiments testing OAS1 localization during viral infections, OAS1 was expressed as described above. At 24 hr post transfection, cells were infected with the indicated virus for 1 hr with gentle rocking followed by removal of the inoculum and replacement with fresh media. At 24 hr post treatment/expression, cells were washed with PBS and then fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 10 min at room temperature, washed with PBS, and then permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature. Cells were washed with PBS and then resuspended in a 3% BSA/PBS blocking solution for 1 hr. After blocking, cells infected with EMCV were stained with rabbit anti-OAS1 (CST) and mouse IgG1 anti-dsRNA 9D5 in PBS containing 1% BSA and 0.3% Triton X-100 for 1 hr in the dark at room temperature. Cells infected with WNV were stained with rabbit anti-OAS1 (CST), mouse IgG1 anti Golgin 97 (CST) or mouse anti IgG1 PDIA3 (Sigma-Aldrich) and mouse IgG2a anti-dsRNA J2 (Scicons) in PBS containing 1% BSA and 0.3% Triton X-100 for 1 hr in the dark at room temperature. EMCV infected 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 hr in the dark at room temperature. 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), goat anti-mouse IgG1 (Thermo Fisher), and goat anti-mouse IgG2a Alexa Fluor 648 (Thermo Fisher) in PBS containing 1% BSA and 0.3% Triton X-100 for 1 hr in the dark at room temperature. Samples were washed once with PBS, stained with DAPI in PBS for 10 min in the dark, followed by washing three times with PBS and then mounted with ProLong Glass antifade mounting media (Thermo Fisher). Samples were cured in the dark at room temperature for 24–48 hr prior to imaging. Samples were imaged using a Nikon Eclipse Ti laser scanning confocal microscope using a 60x oil-immersion lens. Images were processed and analyzed using the NIS elements software and Fiji. Quantification of co-localization was performed using the Fiji Coloc two plugin. RNA isolation, reverse transcription, and RT-qPCR Request a detailed protocol Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg total RNA using the QuantiTect RT kit (Qiagen) according to the manufacturer’s instructions. RT-qPCR was carried out using the ViiA7 RT-qPCR system with TaqMan reagents using TaqMan primers/probes (Life Technologies) for EMCV 5′UTR (Supplementary file 3). RNA immunoprecipitation Request a detailed protocol 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). At 24 hr post transfection, cells were infected with EMCV at an MOI of 0.001. At 12 hr post-infection, cells were harvested and lysed in RNA-IP lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.4, 0.5% NP-40, 1 mM DTT, 1× HALT protease inhibitor, 100 U/mL RNasin and 2 mM ribonucleoside-vanadyl complex). Nuclei and debris were removed from the cytosolic lysate by centrifugation at 8000 g at 4°C for 10 min. Next, 400 μg protein from the cytosolic lysate was incubated with 5 μg anti-FLAG mouse IgG1 (M2, Sigma) or mouse IgG1 control overnight at 4°C, with rotation. The next day, 0.75 mg Dynabeads Protein G (Invitrogen) was added, and the lysate was incubated for 2 hr at 4°C with rotation. After washing, coprecipitated RNA was isolated from IgG1-protein complexes by chloroform-isoamyl alcohol extraction, reverse transcribed into cDNA (QuantiTect RT kit, Qiagen) and analyzed by RT-qPCR. OAS1 in vitro activity assay Request a detailed protocol Enrichment of FLAG-tagged OAS1 isoform proteins prior to in vitro activity assay was performed as described for the RNA-IP above. Briefly, OAS1 KO 293 T cells were seeded on a 10 cm dish and transfected with FLAG-tagged OAS1 isoform expression plasmids and harvested 24 hr post transfection in Co-IP lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% Igepal Ca-630, 1 mM EDTA, 1x HALT protease inhibitor). Lysate was incubated with 10 μg anti-FLAG mouse IgG1 (M2, Sigma) at 4°C, with rotation. The next day, 1.5 mg Dynabeads Protein G (Invitrogen) was added, and the lysate was incubated for 2 hr at 4°C with rotation, and then washed 6x with Co-IP lysis buffer. Immunoprecipitated protein samples were incubated in reaction buffer (10 mM Tris-HCl pH 7.5, 25 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA) supplemented with 400 mM ATP, ~80 nM [a-32P]-labeled ATP (3000 Ci/mmol 10 mCi/mL, 250 µCi; PerkinElmer), and 33.3 µg/mL poly(I:C) (Invivogen). The reactions were allowed to proceed for 2 hr at 37°C. The reactions were then analyzed by denaturing gel electrophoresis on a 20 cm tall 20% polyacrylamide 7 M urea gel with 0.5 x TBE running buffer at 12.5 W. The gels were then applied onto Whatman filter paper, covered with plastic, and exposed directly to a PhosphorImager screen (GE Healthcare) for 15 to 40 min, as necessary. [32P]-labeled 2′−5′ oligoadenylate products were visualized using a Sapphire Biomolecular Imager (Azure Biosystems). Genetic association Request a detailed protocol Samples from a cohort of 34 severe COVID-19 cases were collected starting in April 2020 at Virginia Mason Medical Center and Benaroya Research Institute. Severity was based on hospitalization in the critical care unit with mechanical ventilation. A cohort of 99 healthy control subjects matched for ancestry (self-reported) was assembled from participants in the healthy control registry at Benaroya Research Institute. Both studies were approved by the Institutional Review Board at Benaroya Research Institute (IRB20-036 and IRB07109 respectively). A description of the cohorts is presented in Supplementary file 5. DNA samples from these subjects were genotyped for OAS1 rs10774671 using a Taqman SNP genotyping assay (Thermo Fisher). Genotypes passed Hardy-Weinberg equilibrium analysis. Association testing was performed using gPLINK v2.050 (https://zzz.bwh.harvard.edu/plink/gplink.shtmlI) by logistic regression adjusting for sex and ancestry (race/ethnicity). Replication of genetic association was tested using 1676 critically ill COVID-19 cases collected through the GenOMICC study in the UK and 8380 population-based controls (1:5 cases:controls) from the UK Biobank samples as described (Pairo-Castineira et al., 2021). All subjects were of European descent as determined by ancestry informative markers. DNA was genotyped using the Illumina Global Screening Array v3.0+ multi disease bead chips (Illumina) and subjected to standard quality control filters. Severity of local COVID-19 patients was classified based on maximum disease severity using a 7-point ordinal scale described previously (Cao et al., 2020). 1, not hospitalized with resumption of normal activities; 2, not hospitalized, but unable to resume normal activities; 3, hospitalized, not requiring supplemental oxygen; 4, hospitalized, requiring supplemental oxygen; 5, hospitalized, requiring nasal high-flow oxygen therapy, noninvasive mechanical ventilation, or both; 6, hospitalized, invasive mechanical ventilation; and 7, death. COVID-19 patients with ordinal scores of 6 and 7 were included in the genetic analysis. LD of the two SNPs was assessed from 1000 Genomes http:/
Abstract There is increasing evidence that the complexity of the retinal vasculature measured as fractal dimension, D f , might offer earlier insights into the progression of coronary artery disease (CAD) before traditional biomarkers can be detected. This association could be partly explained by a common genetic basis; however, the genetic component of D f is poorly understood. We present a genome-wide association study (GWAS) of 38,000 individuals with white British ancestry from the UK Biobank aimed to comprehensively study the genetic component of D f and analyse its relationship with CAD. We replicated 5 D f loci and found 4 additional loci with suggestive significance ( P < 1e−05) to contribute to D f variation, which previously were reported in retinal tortuosity and complexity, hypertension, and CAD studies. Significant negative genetic correlation estimates support the inverse relationship between D f and CAD, and between D f and myocardial infarction (MI), one of CAD’s fatal outcomes. Fine-mapping of D f loci revealed Notch signalling regulatory variants supporting a shared mechanism with MI outcomes. We developed a predictive model for MI incident cases, recorded over a 10-year period following clinical and ophthalmic evaluation, combining clinical information, D f , and a CAD polygenic risk score. Internal cross-validation demonstrated a considerable improvement in the area under the curve (AUC) of our predictive model (AUC = 0.770 ± 0.001) when comparing with an established risk model, SCORE, (AUC = 0.741 ± 0.002) and extensions thereof leveraging the PRS (AUC = 0.728 ± 0.001). This evidences that D f provides risk information beyond demographic, lifestyle, and genetic risk factors. Our findings shed new light on the genetic basis of D f , unveiling a common control with MI, and highlighting the benefits of its application in individualised MI risk prediction.
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