Journal: PLoS Pathogens
Article Title: Genome-wide RNAi Screen Reveals a New Role of a WNT/CTNNB1 Signaling Pathway as Negative Regulator of Virus-induced Innate Immune Responses
Figure Lengend Snippet: Genome-wide gene silencing study of virus-induced innate immune responses and bioinformatics analyses. (A) Schematic representation of the primary genome-wide screen and secondary screens. HEK 293T cells stably expressing the luciferase gene under the control of the IFNB1 promoter were transduced with arrayed lentiviruses combining three shRNAs per gene (primary screen) or with five individual shRNA-expressing lentiviruses for each gene hit (secondary screens) in a 96-well format. After 72 hours, cells were challenged with SeV virus (primary and confirmation screens) or transfected with polyinosinic∶polycytidylic acid (polyI∶C), MAVS- or IRF3(5D)-expressing plasmids (secondary screens) for 16 hours before measuring IFNB1 promoter-driven luciferase activity. (B) Decision tree of primary screen and summary data of gene hits obtained in secondary and validation screens. Selected gene hits (114) that were confirmed and validated with endogenous IFNB1 screens by qRT-PCR induced a modulation of more than 25% of the IFNB1 promoter activity with at least two independent shRNAs following SeV infection. Prioritized gene hits (59) for which knockdown of the target gene was greater than 40% with two independent shRNAs are also identified. (C) Schematic representation of confirmation and secondary assays for epistasis analysis of gene hits acting on the signaling cascade leading to IFNB1 production. SeV infection (primary and confirmation screens), polyI∶C (dsRNA mimetic), MAVS or IRF3(5D) expressing plasmids transfection (secondary screens) were used to activate innate immune response. A non-specific assay was used to discard gene hits affecting nonimmune-related transcription by measuring transcriptional activity of EF1α constitutive promoter. (D) Heat map indicating modulation of IFNB1 promoter activity following silencing of control genes in confirmation and secondary assays (log 2 scale). The functional profiling data for controls and gene hits allow classification within four functional groups: I - SeV specific, II - cytoplasmic dsRNA sensing, III - MAVS-dependent signaling, IV - nuclear import or transcription factor-dependent process. (E) Functional profiling data of 114 gene hits confirmed with at least two shRNAs in the SeV confirmation screen and further validated using endogenous IFNB1 mRNA quantification by qRT-PCR. Manual clustering was performed to classify each gene hit in one of the four functional groups. (F) qRT-PCR validation data of the endogenous IFNB1 mRNA levels and target gene knockdown efficiency in transduced cells with each lentivirus-expressing shRNA for the 114 gene hits. (G) Enriched Gene Ontology (GO) biological process and molecular function terms (P
Article Snippet: Large-scale lentiviral shRNA production 293T cells were transfected using PEI with 6 µg pLKO.1-puro encoding shRNA targeting WNT2B (TRCN0000033376 and TRCN0000033377), WNT9B (TRCN0000062069 and TRCN0000062070), WLS (TRCN0000133858 and TRCN0000133999), CTNNB1 (TRCN0000003843, TRCN0000003844 and TRCN0000003845) or shRNA non-target (NT, Sigma), 1.5 µg pMDLg/pRRE, 1.5 µg pRSV-REV and 3 µg pVSVg as previously described .
Techniques: Genome Wide, Stable Transfection, Expressing, Luciferase, Transduction, shRNA, Transfection, Activity Assay, Quantitative RT-PCR, Infection, Functional Assay