Additional file 1: of Correction to: Dual RNA-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome

Table S2. Gene count table for 48 RNA-seq nasal brushing subjects from GALA cohort. (XLSX 5732 kb

Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

<div><p>Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref001" target="_blank">1</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref004" target="_blank">4</a>]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.</p></div

Additional file 1: of Dual RNA-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome

Table S1. Clinical characteristics of subjects. Table S2. Gene count table for 48 RNA-seq nasal brushing subjects from GALA cohort. Table S3. Virus-High DESeq differential expression analysis. Table S4. DAVID enrichment for Virus High DEGs. Table S5. Gene count table for HRV in vitro stimulation dataset. Table S6. HRV in vitro DEGs. Table S7. Virus Low Differential Expression Analysis. Table S8. WGCNA gene2module. Table S9. DAVId for modules. Table S10. immune cell enrichments for modules. (XLS 18739 kb

Additional file 2: Figure S1. of Dual RNA-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome

Study design. Figure S2. Genome coverage for viruses detected in Ion Torrent Proton sequenced samples. Figure S3. Comparison of in vivo and in vitro log2 gene expression fold changes by infection status. Figure S4. WGCNA gene correlation dendrogram showing nine detected gene co-expression modules. Figure S5. H&E staining of nasal airway epithelial brushing cell cytospin from a viral-infected subject. (PDF 30208 kb

IRF3 is Required for G10-Dependent Transcription and Anti-Alphaviral Activity.

<p>(A) Immunoblot showing IRF3, STAT1, and GAPDH in THF-ISRE stably transduced with Cas9 and CRISPR gRNA directed against either STAT1 (THF-ISRE-ΔSTAT1) or IRF3 (THF-ISRE-ΔIRF3) as indicated. (B) Induction of IRF3/IFN-dependent LUC in THF lacking STAT1 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are average fold changes ±SD of quadruplicate measurements relative to cells exposed only to 1% DMSO. (C) Induction of IRF3/IFN-dependent LUC in THF lacking IRF3 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are as in B. (D) Immunoblot of lysates from THF-ISRE following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (E) Average media titers +SD of CHIKV, VEEV, and SINV at 24 (VEEV) or 48hpi (CHIKV, SINV) obtained from THF-ISRE-ΔIRF3 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

G10 Elicits IRF3 phosphorylation and Anti-Alphaviral Activity in Cells Lacking IPS1.

<p>(A) Immunoblot of lysates from THF-ISRE-ΔIPS1 following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Average media titers +SD of CHIKV, VEEV, and SINV at 24h (VEEV) or 48h (CHIKV, SINV) post infection obtained from THF-ISRE-ΔIPS1 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

G10-Mediated IRF3 Phosphorylation, ISG mRNA Induction, and Anti-Alphaviral Activity are not Detectable in Cells Lacking STING.

<p>(A) Immunoblot of lysates from THF-ΔSTING following 7h exposure to 1% DMSO, 0.1μg/mL poly(I:C), SeV, UV-CMV, 1μg/mL 2’3’-cGAMP, or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Expression of IRF3/IFN-dependent LUC in THF-ISRE-ΔIPS1 and THF-ISRE-ΔSTING following 7h exposure to 1% DMSO, indicated concentrations of G10, SeV, UV-CMV, or 1μg/mL LPS. Values are presented as average fold change in quadruplicate measurements ±SD relative to cells treated with 1% DMSO. (C) Average fold changes ±SD from duplicate experiments of ISG54, ISG15, and Viperin mRNA relative to cells treated with 1% DMSO in THF-ISRE-ΔIPS1 (black bars) or THF-ISRE-ΔSTING (gray bars) following exposure to UV-CMV, SeV, or 100μM G10. (D) Media titers of CHIKV and VEEV at 24h (VEEV) or 48h (CHIKV) obtained from THF-ISRE-ΔSTING cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

Comparative Kinetics and Dose-Dependence of Innate Immune Activation by G10 and 2’3’-cGAMP.

<p>(A) Immunoblot of lysates from THF-ISRE cells following exposure to G10 (100μM) or transfected 2’3’-cGAMP (42.3μM) for indicated time showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (B) mRNA synthesis of indicated genes in THF following 8h exposure to indicated concentration of G10 (blue) or 2’3’-cGAMP (red). Indicated values represent average mRNA fold change ±SD from duplicate experiments relative to cells exposed to 1% DMSO.</p

G10 Elicits Antiviral Activity.

<p>Average titers ±SD of VACV, WNV, CHIKV, and VEEV grown on THF cells in the presence of indicated G10 concentration (DMSO concentration normalized to 1%). Infections were performed in triplicate and virus harvested at 48h post infection (CHIKV, WNV, CHIKV) or 24h post infection (VACV, VEEV).</p

G10 Induces IFN/IRF3- but not NF-κB-Dependent Transcription in Human Fibroblasts.

<p>(A) Dose-dependent expression of IRF3/IFN-dependent luciferase (LUC) in telomerized human fibroblasts (THF). Values displayed are average fold changes ±SD of quadruplicate measurements of luminescence following 7h exposure to indicated concentration of G10 relative to cells exposed only to 1% DMSO (all samples normalized to 1% DMSO). (B) mRNA transcription of genes dependent on IRF3/IFN following 7h exposure to 100μM G10 or UV-CMV. Indicated values represent average ±SEM mRNA fold change relative to cells exposed to 1% DMSO from duplicate experiments. (C) Induction of NF-κB-dependent LUC signal in THF reporter cells following 7h exposure to 1μg/mL LPS, 160 HA units/mL SeV, 1ng/mL TNFα or indicated concentration of G10. Values displayed are as described in (A). (D) mRNA transcription of of NF-κB-dependent genes following 7h exposure to 100μM G10 or 160 HA units/mL SeV. Indicated values are mRNA fold change relative to cells exposed to 1% DMSO and are representative of duplicate experiments.</p