Comment[ArrayExpressAccession] E-GEOD-60573 MAGE-TAB Version 1.1 Public Release Date 2014-08-21 Investigation Title Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity [Kinetic] Comment[Submitted Name] Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity [Kinetic] Experiment Description The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5M-bM-^@M-^Y triphosphate (5M-bM-^@M-^Yppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5M-bM-^@M-^YpppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, andinduction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN)signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5M-bM-^@M-^YpppRNA, and not by IFNM-NM-1-2bthat included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5M-bM-^@M-^YpppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5M-bM-^@M-^YpppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach providestranscriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5M-bM-^@M-^YpppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents. Kinetic analysis of A549 cells treated with 5'pppRNA and analyzed at 1h, 2h, 4h, 6h, 8h, 12h, 24h or 48h. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Hiscott Goulet Olagnier Xu Paz Belgnaoui Lafferty Janelle Arguello Paquet Ghneim Richards Smith Wilkinson Cameron Kalinke Qureshi Lamarre Haddad Sekaly Peri Balachandran Lin Hiscott Person First Name John Marie-Line David Zhengyun Suzanne S Erin ValM-CM-)rie Meztli Marilene Khader Stephanie Andrew Peter Mark Ulrich Salman Alain Elias Rafick Suraj Siddharth Rongtuan John Person Mid Initials M I K P Person Email jhiscott@vgtifl.org Person Affiliation Vaccine and Gene Institute-Florida Person Address Infectious Diseases, Vaccine and Gene Institute-Florida, 9801 SW Discovery Way, Port Saint Lucie, Florida, USA Person Roles submitter Protocol Name P-GSE60573-1 P-GSE60573-5 P-GSE60573-6 P-GSE60573-2 P-GSE60573-3 P-GSE60573-4 P-GSE60573-7 Protocol Description Analysis of the GenomeStudio output data was conducted using the R statistical language and various software packages from Bioconductor by the Bioinformatics core at the Collaborative Genomics Center. Missing values were imputed using the KNN algorithm from the impute R package. Quantile normalization was applied, followed by a log2 transformation. ID_REF = VALUE = Quantile normalized, log2 transformed intensities The hybridized BeadChips were washed, blocked, stained and scanned according to the Illumina's direct hybridization protocol. Microarray analysis was conducted using 750ng of biotinylated cRNA hybridized to Human RefSeq-12 V4 BeadChips (Illumina) at 58M-0C for 20 hours, according to Illumina's direct hybridization protocol by the Genomics core at Collaborative Genomics Center (CGC). Cells were either transfected with 5'pppRNA (10ng/mL) using RNAiMax and collected at 1h, 2h, 4h, 6h, 8h, 12h, 24h or 48h post-treatment, or left non-treated and collected at 6h A549 cells were cultured as per ATCC recommendations. RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser. The arrays were scanned using an Illumina iScan scanner and quantified using GenomeStudio software (Illumina). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATED WITH TIME Experimental Factor Type treated with time Publication Title Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity. Publication Author List Goulet ML, Olagnier D, Xu Z, Paz S, Belgnaoui SM, Lafferty EI, Janelle V, Arguello M, Paquet M, Ghneim K, Richards S, Smith A, Wilkinson P, Cameron M, Kalinke U, Qureshi S, Lamarre A, Haddad EK, Sekaly RP, Peri S, Balachandran S, Lin R, Hiscott J PubMed ID 23633948 Publication DOI 10.1371/journal.ppat.1003298 Comment[SecondaryAccession] GSE60573 Comment[GEOReleaseDate] 2014-08-21 Comment[ArrayExpressSubmissionDate] 2014-08-20 Comment[GEOLastUpdateDate] 2014-08-21 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE60573_non_normalized.txt SDRF File E-GEOD-60573.sdrf.txt