Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 1. 59pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells. (A) Schematic representation of VSV-derived 59pppRNA and gel analysis. The 59ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of in vitro transcription runs as a single product degraded by RNase I. (B) 59pppRNA or a homologous control RNA lacking a 59- triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and b-actin. Results are from a representative experiment; all immunoblots are from the same samples. (C) A549 cells were transfected with 10 ng/ml 59pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IkBa pSer-32, IkBa and b-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. (D) ELISA was performed on cell culture supernatants to quantify the release of IFNb and IFNa over time. Error bars represent SEM from two independent samples. doi:10.1371/journal.ppat.1003298.g001

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: Derivative Assay, In Vitro, Control, Transfection, SDS Page, Western Blot, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 2. Induction of the interferon response by 59pppRNA is dependent on functional RIG-I signaling. (A) WT and RIG-I2/2 MEFs were co-transfected with IFNa4 or IFNb promoter reporter plasmid (200 ng) along with 59pppRNA (500 ng/ml) or expression plasmids encoding a constitutively active form of RIG-I (DRIG-I) (100 ng). IRF-7 expression plasmid (100 ng) was added for transactivation of the IFNa4 promoter. Luciferase activity was analyzed 24 h post-transfection by the Dual-Luciferase Reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Error bars represent SEM from nine replicates performed in three independent experiments. (B) Mda52/2, TLR32/2, TLR72/2 and RIG-I2/2 MEFs were co-transfected with IFNb promoter reporter plasmid (200 ng) along with 59pppRNA (500 ng/ml). Luciferase activity was analyzed 24 h post-transfection by the Dual-Luciferase Reporter assay. Relative luciferase activity was measured as fold induction relative to the basal level of reporter gene. Promoter activity in the knockout MEFs was then normalized against the activity in their respective wt MEFs to obtain the percentage of fold activation. Error bars represent SEM from nine replicates performed in three independent experiments. (C) A549 cells were either left untreated or transfected with a control siRNA or RIG-I siRNA. After 48 h, 59pppRNA (10 ng/mL) was transfected and at 8 h after treatment, WCEs were analyzed by SDS-PAGE and immunoblotted for pIRF3 Ser-396, IRF3, pSTAT1 Tyr 701, STAT1, IFIT1, RIG-I, and b-Actin. Results are from a representative experiment; all immunoblots are from the same samples. doi:10.1371/journal.ppat.1003298.g002

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Reporter Assay, Knock-Out, Activation Assay, Control, SDS Page, Western Blot

Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 3. Transcriptome analysis of the host antiviral response to 59pppRNA. A549 cells were transfected with 10 ng/ml of 59pppRNA using Lipofectamine RNAiMax for designated periods of time. Samples were analyzed by Illumina gene expression array and DEG were identified based on fold change $62 and p-value #0.001 (A) Number of up-regulated and down-regulated DEG at each time point. (B) Heatmap of all DEG sorted by fold change; top 30 genes are listed. Red, Up-regulated; blue, down-regulated. (C) Functional characterization of DEGs following 59pppRNA treatment based on Ingenuity Pathway Analysis software. Bar height refers to the number of DEG in each pathway and the color refers to the contribution from up-regulated or down-regulated genes. (D) Genes among the top up-regulated genes were selected based on three different expression patterns: early, sustained, late. doi:10.1371/journal.ppat.1003298.g003

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: Transfection, Gene Expression, Functional Assay, Software, Expressing

Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 5. Gene expression profiling of differentially expressed genes in response to 59pppRNA and IFNa-2b. A549 cells were transfected with 10 ng/ml of 59pppRNA using Lipofectamine RNAiMax or treated with IFNa-2b (100 IU/ml or 1000 IU/ml). Samples were collected at 6 h or 24 h post-treatment and were analyzed by Illumina gene expression array. Genes with a fold change $62.0 and p-value #0.001 were considered differentially expressed. (A) Heatmap showing top DEG affected by 59pppRNA and IFNa-2b treatments. Genes regulated by both 59pppRNA and IFNa-2b in at least one condition are indicated in black (up-regulated) or blue (down-regulated). Genes uniquely induced by 59pppRNA in at least one time point but not by IFNa-2b in any conditions are highlighted in red (up-regulated) or green (down-regulated). Top genes are listed in each instance. (B) Cell culture supernatant was collected at the time of treatment with IFNa-2b (0 h; input), or at 24 h following 59pppRNA or IFNa-2b treatment and assayed by ELISA for multiple subunits of IFNa. Error bars represent SEM from two independent samples. (C) Surface proportional Venn diagram illustrating the magnitude of the response by 59pppRNA and IFNa-2b at 6 h and 24 h. Number of DEG is indicated in each area. (D) Comparison of genes induced by each treatment - 59pppRNA 6 h (dark blue); 59pppRNA 24 h (light blue); IFNa-2b (1000 IU/ml) 6 h (red); IFNa-2b (1000 IU/ml) 24 h (pink) - based on functional classification by Ingenuity Pathway Analysis. doi:10.1371/journal.ppat.1003298.g005

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: Gene Expression, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Functional Assay

Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 6. 59pppRNA acts as a broad-spectrum antiviral agent. (A) A549 cells were transfected with 10 ng/ml 59pppRNA 24 h prior to infection with VSVD51-GFP (MOI 0.1), Dengue virus (MOI 0.1), and Vaccinia-GFP virus (MOI 5), respectively. Percentage of infected cells was determined 24 h post-infection by flow cytometry analysis of GFP expression (VSV-GFP and Vaccinia-GFP) or intracellular staining of DENV E protein expression (Dengue virus). Data are from a representative experiment performed in triplicate 6 SD. (B) Human PBMCs were transfected with 100 ng/ ml 59pppRNA 24 h prior to infection with dengue virus at an MOI of 5. At 24 h post-infection, the percentage of Dengue infected CD14+ and CD142 cells was evaluated by intracellular staining of DENV E protein expression by flow cytometry. Data are from a representative experiment performed in triplicate 6 SD. (C) Human PBMCs from three different donors were transfected with 100 ng/ml 59pppRNA prior to infection with Dengue virus at an MOI of 5. The percentage of Dengue infected cells in the CD14+ population was evaluated by intracellular staining of DENV E protein expression using flow cytometry. Data are from an experiment performed in triplicate on three different patients 6 SD (D) CD4+ T cells isolated from human PBMCs and activated with anti-CD3 and anti-CD28 antibodies. Cells were incubated in the presence or absence of supernatant from 59pppRNA- treated monocytes for 4 h and infected with HIV-GFP (MOI 0.1) for 48 h. The percentage of HIV infected, activated CD4+ T cells (GFP positive) was assessed by flow cytometry. (E) Huh7 and Huh7.5 were transfected with 59pppRNA (10 ng/mL) and infected with HCV 24 h later. At 48 h post- infection, WCEs were collected and subjected to SDS PAGE and immunoblot to examine the expression of HCV viral protein NS3, IFIT1, and b-Actin. doi:10.1371/journal.ppat.1003298.g006

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: Transfection, Infection, Virus, Flow Cytometry, Expressing, Staining, Isolation, Incubation, SDS Page, Western Blot

Journal: PLoS pathogens

Article Title: Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

doi: 10.1371/journal.ppat.1003298

Figure Lengend Snippet: Figure 7. 59pppRNA inhibits H1N1 Influenza replication in vitro. (A) A549 cells were treated with 59pppRNA (10 ng/ml); at 24 h post- treatment, cells were infected with an increasing MOI of A/PR8/34 H1N1 Influenza virus (0.02MOI, 0.2MOI, 2MOI) for 24 h. WCEs were subjected to SDS PAGE and immunoblot to examine the expression of influenza viral protein NS1, ISG56, and b-Actin. (B) Viral titers in cell culture supernatants from (A) were determined by plaque assay. Error bars represent SEM from two independent samples. (C) A549 cells were pre-treated with increasing concentrations of 59pppRNA (0.1 ng/ml to 10 ng/ml) for 24 h, prior to 0.2MOI influenza challenge. WCEs were examined for influenza NS1, ISG56, b- Actin by immunoblot. (D) Viral titers in cell culture supernatants from (C) were determined by plaque assay. Error bars represent SEM from two independent samples. (E) A549 cells were transfected with a control siRNA, RIG-I siRNA or IFNa/bR siRNA and then treated with 59pppRNA (10 ng/ml), followed by infection with Influenza (0.2MOI). WCEs were prepared 24 h later and subjected to SDS PAGE and immunoblot to examine the expression of influenza NS1, RIG-I, IFIT1, and b-Actin. (F) A549 cells were transfected with a control siRNA or IFNa/bR siRNA and then treated with 59pppRNA (10 ng/ml) or IFNa-2b (100 IU/mL) for 24 h. Expression of IFIT1, RIG-I and b-Actin was evaluated by western blotting. doi:10.1371/journal.ppat.1003298.g007

Article Snippet: For siRNA knock down, A549 cells were transfected with 50 nM (30 pmol) of human RIG-I (sc-61480), IFN-a/bR a (sc-35637) and b (sc-40091) chain, or control siRNA (sc-37007) (Santa Cruz Biotechnologies, Dallas, USA) using Lipofectamine RNAi Max (Invitrogen, NY, USA) according to the manufacturer’s guidelines.

Techniques: In Vitro, Infection, Virus, SDS Page, Western Blot, Expressing, Cell Culture, Plaque Assay, Transfection, Control

Journal: Cell reports

Article Title: Molecular basis of autoimmune disease protection by MDA5 variants.

doi: 10.1016/j.celrep.2025.115754

Figure Lengend Snippet: Figure 1. Effects of T1D-protective MDA5 variants on the antiviral interferon response and the ATPase and filament-forming activities of MDA5 (A) MDA5 domain organization. CARD, caspase recruitment domain; Hel1 and Hel2, RecA-like helicase domains; Hel2i, Hel2 insert domain; P, pincer domain; CTD, C-terminal domain. (B) Western blot showing doxycycline-inducible expression of human MDA5 variants in stable A549 RIG-I KO cell lines and the absence of endogenous MDA5 expression in the parent RIG-I KO cell line. (C) Western blot showing doxycycline-induced expression of MDA5 variants in the A549 cell lines after poly(I:C) stimulation. (D) RT-qPCR quantification of IFNB1 and IFIT1 mRNA in A549 RIG-I KO cells stably expressing the indicated human MDA5 variant under a doxycycline-inducible promoter 7 h after infection with encephalomyocarditis virus (EMCV). I923V and E627* inhibit the antiviral response. hRef, human reference sequence. Note that hRef MDA5 has H843; mouse MDA5 has R843. (E) RT-qPCR quantification of EMCV RNA 7 h post-infection. See also Figure S1. Error bars in (D) and (E) represent mean ± SEM (2 or 3 measurements from 3 independent experiments).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Atomic model: mMDA5 I923V (no nucelotide) This paper PDB: 9F0J Atomic model: mMDA5 A946T (no nucelotide) This paper PDB: 9F3P Experimental models: Cell lines Human: HEK293T cells ATCC RRID: CVCL_0063 Hamster: BHK-21 fibroblasts ATCC RRID: CVCL_1915 Human: A549 RIG-I − /− cells Teague et al. 59 N/A Human: A549 RIG-I − /− ACE2 + cells This paper N/A Recombinant DNA Plasmid: pLVX-TetOne-Puro Takara Cat#631849 Plasmid: pLVX-TetOne-Puro-hMDA5 This paper N/A Plasmid: pET28a(+) SigmaAldrich Cat#69864 Software and algorithms Octet Analysis Studio v11.1 Sartorius sartorius.com Prism v10 GraphPad graphpad.com PR.Stability Analysis v1.1 NanoTemper nanotempertech.com EPU ThermoFisher thermofisher.com SerialEM Mastronarde et al. 67 bio3d.colorado.edu MotionCor2.0 Kimanius et al. 68 N/A Relion4.0 He et al. 70 relion.readthedocs.io CtfFind4.1 Rohou et al. 69 N/A crYOLO Wagner et al. 71 cryolo.readthedocs.io UCSF Chimera Pettersen et al. 73 www.cgl.ucsf.edu/chimera COOT Emsley et al. 74 www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot Phenix 1.21 Liebschner et al. 75 www.phenix-online.org PISA server Krissinel et al. 61 ebi.ac.uk/pdbe/prot_int Fiji v2.16 Schindelin et al. 66 https://fiji.sc Other QUANTIFOIL R1.2/1.3 300-mesh gold grids Agar Scientific Cat#AGS143-8 HisTrap HP nickel-affinity column Cytiva Cat#17-5248-02 Octet SA Biosensors Sartorius Cat#18-5019 Standard capillaries NanoTemper Cat#PR-C002 14 Cell Reports 44, 115754, June 24, 2025

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Stable Transfection, Variant Assay, Infection, Virus, Sequencing