Figure 1.
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 (log2 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<0.05) for the 114 genes confirmed by qRT-PCR relative to all genes examined in the genome-wide RNAi screen. (H) Identification of 17 out of 237 gene hits that are induced by more than twofold after SeV infection or IFN-α treatment in our microarray analysis of HEK 293T cells (13 genes) or previously described as ISGs (4 genes in gray) [75]. The gene hits that modulate endogenous IFNB1 mRNA are indicated in bold. (I) Venn diagram representing the overlap between various RNAi screens including: our RLR RNAi screen (290 genes validated in secondary screens, including 53 hits considered as non specific in pEF1α assay), a NOD-like receptor screen (NLR) and nine independent viral replication screens (HIV, influenza, HCV and West Nile virus). Numbers between brackets represent the overlap when only the 237 genes considered as specific in our RLR screen are included.
Figure 2.
WNT2B and WNT9B ligands as novel negative regulators of antiviral innate immunity.
(A) Heat map indicating log2 effect of silencing WNT2B and WNT9B genes with 3 independent lentivirus-expressing shRNAs (left) and qRT-PCR validation of endogenous IFNB1 mRNA levels and of target gene knockdown efficiency (right). (B) Immunoblot analysis of IFIT1, IFIT2 and DDX58 in WNT2B, WNT9B and WLS knockdown HEK 293T cells (two-independent shRNAs per gene) following infection with SeV for 16 hours. (C) IFNB1 promoter-driven luciferase activity in HEK 293T cells treated as described in (B). (D) Fold induction of IFIT1 mRNA levels in HEK 293T cells treated as described in (B). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) ELISA quantification of secreted IFN-β protein in supernatant of HEK 293T cells treated as described in (B). (F) Immunoblot analysis of HEK 293T supernatants and lysates at various time points in cells overexpressing WNT2B and WNT9B and infected with SeV or treated with IFN-α. The specific bands are indicated with arrows. (G) Fold induction of CTNNB1-TCF/LEF dependent reporter activity (TOP-flash) with addition of purified WNT3A or WNT9B proteins to HEK 293T cell supernatants for 16 hours. (H) Relative fold induction of IFNB1- or EF1α-driven reporter activity with dose-dependent addition of purified WNT9B protein to cell supernatants of SeV infected HEK 293T cells for 16 hours. (I–J) Fold induction of IFNB1 (I) or IFIT1 (J) mRNA levels following dose-dependent addition of purified WNT3A or WNT9B proteins to cell supernatants of SeV infected HEK 293T cells for 16 hours. qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. P values<0.05 (*), <0.01 (**) or <0.001 (***) are indicated.
Figure 3.
CTNNB1 is a negative regulator of antiviral innate immunity.
(A) IFNB1 promoter-driven luciferase activity in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or 3 shRNAs targeting CTNNB1 for four days and subjected to SeV infection for 16 hours. (B) Immunoblot analysis of CTNNB1 and IFIT1 in HEK 293T cells transduced with 3 lentivirus-expressing shRNAs targeting CTNNB1 and infected with SeV. (C) Immunoblot analysis of CTNNB1, IFIT1 and DDX58 in SeV-infected HEK 293T cells previously transduced with a shRNA or transfected with a pool of four siRNAs targeting CTNNB1 for three days. (D–F) Fold induction of IFNB1 (D), DDX58 (E) and TNF (F) mRNA levels in HEK 293T cells treated as described in (C). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (G) ELISA quantification of secreted IFN-β protein in supernatant of HEK 293T cells treated as described in (A). (H–I) Fold induction of IFNB1 promoter-driven luciferase activity (H) and of CTNNB1-TCF/LEF dependent reporter activity (TOP-flash, I) following dose-dependent transfection of CTNNB1-expressing plasmid (50, 100, 150 and 200 ng) for 48 hours in SeV-infected HEK 293T cells.
Figure 4.
SeV infection induces CTNNB1 stabilization and nuclear translocation.
(A) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, CTNNB1 phosphorylated at Ser552, NFKBIA (IκBα) phosphorylated at Ser32, IFIT1, DDX58 and SeV protein HN of HEK 293T transduced with lentivirus-expressing shRNA 45 targeting CTNNB1 and shRNA NT (control) for four days and subjected to SeV infection for 0, 4, 8, 24, 32 or 48 hours. (B–D) Fold induction of IFNB1 (B), IFIT1 (C) and TNF (D) mRNA levels in HEK 293T cells treated as described in (A). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Confocal analysis of HEK 293T cells using Hoechst, anti-CTNNB1 active form and anti-IRF3 antibodies without virus infection or following 16 hours infection with SeV. Nuclear detection of IRF3 and CTNNB1 is identified by white arrows in infected cells.
Figure 5.
Pharmacological inhibition of GSK3 stabilizes an active CTNNB1 pool and negatively regulates antiviral innate immunity.
(A–B) Relative fold induction of IFNB1-driven reporter activity (A) and fold induction of CTNNB1-TCF/LEF dependent reporter activity TOP-flash (B) in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or shRNA 45 targeting CTNNB1 for four days and subjected to GSK3 inhibition with BIO (5 µM) or BIO-acetoxime (10 µM) and SeV infection for 16 hours. (C) Immunoblot analysis of autophosphorylated GSK3α/β at Tyr279/216, dephosphorylated active CTNNB1 at Ser37/Thr41 and IFIT1 in HEK 293T cells treated with GSK3 inhibitors BIO (5 µM) or BIO-acetoxime (10 µM) and infected with SeV for 16 hours. (D) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, IFIT1 and phosphorylated NFKBIA (IκBα) at Ser32 in SeV-infected HEK 293T cells treated as described in (A–B) and treated with BIO (5 µM) or BIO-acetoxime (10 µM). (E) Relative fold induction of TOP-flash, IFNB1, ISG56, NF-κB and EF1α-driven reporter activity in HEK 293T cells subjected to GSK3 inhibition with BIO (5 µM), TCF/LEF interaction inhibition with PNU74654 (6 µM) or both inhibitors prior to SeV infection for 16 hours.
Figure 6.
WNT/CTNNB1 signaling acts as a negative regulator of the innate immune response in primary human cells.
(A–B) Fold induction of IFNB1 (A) and TNF (B) mRNA levels in NHBE cells transduced with lentivirus-expressing shRNA 77 targeting WNT2B, shRNA 69 targeting WNT9B, shRNA 58 targeting WLS, shRNA 45 targeting CTNNB1 and NT shRNA in control cells for four days and infected with SeV for 16 hours. (C) Fold induction of IFNB1, IFIT1 and TNF mRNA levels in human monocyte-derived macrophages subjected to GSK3 inhibition with BIO (5 µM) and SeV infection for 4 hours. qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (D) Fold induction of IFIT1 mRNA in primary human hepatocytes transduced with lentivirus-expressing shRNA NT or shRNA 45 targeting CTNNB1 for four days and subjected to GSK3 inhibition with BIO (5 µM) and SeV infection for 5 hours. qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, IFIT1 and HCV NS3 protease in primary human hepatocytes either transduced with lentivirus-expressing shRNA 45 targeting CTNNB1 for four days and/or subjected to GSK3 inhibition with BIO (5 µM) and infected with Hepatitis C virus (HCV) strain JFH1 for 48 hours. (F) Huh7 cells transduced with shRNAs NT, WNT2B 76, WNT2B 77, WNT9B 69, WNT9B 70, WLS 58, WLS 99, CTNNB1 45 or YB1 48 (positive control) were infected with HCV J6/JFH-1(p7-Rluc2A) reporter virus. Following 96 hours of infection, Rluc was measure to assess HCV viral replication and results are represented as relative viral infection with shRNA NT treated cells arbitrarily set to 1. P values<0.05 (*), <0.01 (**) or <0.001 (***) are indicated.
Figure 7.
CTNNB1 associates with IRF3 and NF-κB subunit p65.
HEK 293T cells were transfected with FLAG-MCS (control), FLAG-eYFP (control), FLAG-IRF3 or FLAG-p65 expressing plasmids for forty-eight hours. Cell extracts were prepared following 16 hours of treatment with IFN-α, GSK3 inhibitor BIO (5 µM) or SeV infection before being subjected to immunoprecipitation directed against FLAG. Cell extracts and immune complexes were analyzed by Western blotting using anti-FLAG and anti-CTNNB1 antibodies.
Figure 8.
Cellular map of prioritized gene hits as potential modulators of innate immunity.
Cellular mapping of gene products of 96 gene hits out of 114 that modulated endogenous IFNB1 expression either as Positive Regulators (71 PRs: Red) or as Negative Regulators (25 NRs: green) of the signaling pathway following SeV infection. PRs and NRs were positioned on a cell map and classified according to sub-cellular compartments and cellular function using annotation information from Gene Ontology, KEGG pathway, Uniprot, NCBI and Ingenuity pathway Analysis (IPA). The four functional groups are represented by different symbols: I - SeV specific (triangle), II - cytoplasmic dsRNA sensing (diamond), III - MAVS-dependent signaling (square) and IV - Nuclear import or transcription factor-dependent process (circle). Gene hits with knockdown efficiency confirmed by qRT-PCR with at least two independent shRNAs are highlighted in bold (41 PRs and 9NRs). The RLR and WNT signaling pathways are represented in light and dark blue respectively. Virus-induced activation of the WNT/CTNNB1 canonical-like pathway in feedback inhibition of antiviral innate responses is indicated in red.